Sequence-specific binding polymers for duplex nucleic acids

ABSTRACT

The present invention describes a polymer composition effective to bind, in a sequence-specific manner to a target sequence of a duplex polynucleotide, at least two different-oriented Watson/Crick base-pairs at selected positions in the target sequence. The composition includes an uncharged backbone with 5- or 6-membered cyclic backbone structures and selected bases attached to the backbone structures effective to hydrogen bond specifically with different oriented base-pairs in the target sequence. Also disclosed are subunits useful for the construction of the polymer composition. The present invention further includes methods for (i) coupling a first free or polymer-terminal subunit, and (ii) isolating, from a liquid sample, a target duplex nucleic acid fragment having a selected sequence of base-pairs.

The present invention is a continuation-in-part application of U.S.patent application Ser. No. 719,732, filed Jun. 20, 1991, now U.S. Pat.No. 5,166,315 and herein incorporated by reference, which is acontinuation-in-part of U.S. patent application Ser. No. 07/454,055,filed Dec. 20, 1989 (now issued as U.S. Pat. No. 5,034,506).

FIELD OF THE INVENTION

The present invention relates to an uncharged polymer capable of bindingwith sequence specificity to double stranded nucleic acids containing aselected base-pair sequence.

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BACKGROUND OF THE INVENTION

Oligonucleotides or oligonucleotide analogs designed to inactivateselected single-stranded genetic sequences unique to a target pathogenwere first reported in the late 1960's by Belikova, 1967, andsubsequently by: Pitha, 1970; Summerton, 1978a,b, 1979a,b; Zamecnik,1978; Jones, 1979; Karpova, 1980; Miller, 1979, 1980, 1985; Toulme,1986; and Stirchak, 1987, 1989. Polymeric agents of this type achievetheir sequence specificity by exploiting Watson/Crick base pairingbetween the agent and its complementary single-stranded target geneticsequence. Because such polymers only bind single-stranded target geneticsequences, they are of limited value where the genetic information onewishes to inactivate exists predominantly in the double-stranded state.

For many pathogens and pathogenic states duplex genetic sequences offera more suitable target for blocking genetic activity. One of theearliest attempts to develop a sequence-specific duplex-directed nucleicacid binding agent was reported by Kundu, Heidelberger, and coworkersduring the period 1974 to 1980 (Kundu 1974; Kundu 1975; Kundu 1980).This group reported two monomeric agents, each designed to hydrogen-bondto a specific base-pair in duplex nucleic acids. However, these agentswere ineffective, probably for two reasons. First, they utilized anonrigid ambiguous hydrogen-bonding group (an amide) which can act aseither a proton donor or acceptor (in the hydrogen-bonding sense).Secondly, they provided an insufficient number of hydrogen bonds (two)for complex stability in aqueous solution. Experimental results from avariety of systems suggest that hydrogen-bonded complexes are stable inaqueous solution only if there are a substantial number (probably atleast 12) of cooperative intermolecular hydrogen bonds, or if there areadditional stabilizing interactions (electrostatic, hydrophobic, etc.).

Another early attempt was reported by Dattagupta and Crothers at Yaleand coworkers in Germany (Kosturko 1979; Bunemann 1981). These workersemployed a polymer prepared from a dye known to intercalate into duplexDNA rich in G:C base-pairs and another dye which preferentially binds toduplex DNA rich in A:T base-pairs, probably via minor-groove sites.Preparation of the polymer involved modification of the two dyes byadding acrylic moieties and then polymerization of a mixture of themodified dyes in the presence of duplex DNA of defined sequence (thetemplate). The expectation was that the resultant polymer would show aspecific affinity for duplex DNA having the same sequence as thetemplate DNA. However, such material proved to exhibit only nominalsequence specificity. A variety of bis-intercalating agents designed tobind to specific sequences in duplex DNA have also been reported(Pelaprat, 1980), but such agents inherently give only minimal sequencespecificity.

More recently, Dervan has taken a natural B-form-specificminor-groove-binding antibiotic (Distamycin) and systematically extendedits structure to achieve a significant level of sequence specificity(Schultz 1982; Schultz 1983; Youngquist 1985). He has also appended tothis oligomer an EDTA/Fe complex which under certain conditions acts tocleave the duplex target sequence near the agent's binding site.However, this particular approach will not lead to the high level ofspecificity which is needed for therapeutic applications because theinherent symmetry of the H-bonding sites in the minor groove providestoo little sequence information.

Still more recently, Dervan and coworkers reported a binding agent whichutilizes the informationally-richer polar major-groove sites of a targetgenetic duplex for sequence-specific recognition (Sluka 1987). Thisentailed adapting a synthetic polypeptide, comprising theDNA-sequence-recognition portion of a DNA-binding protein, for cleavingDNA at the protein's binding site on duplex DNA. The cleaving activitywas achieved by linking an EDTA/Fe complex to the amino terminus of thesynthetic peptide and demonstrating that this complex selectivelycleaved duplex DNA at or near the parent protein's natural targetsequence.

Another approach to duplex targeting has grown out of studies firstreported in the late 1950's that demonstrated, via X-ray diffraction,that under high salt conditions an all-thymine or all-uracilpolynucleotide can bind to specific polar major-groove sites on aWatson/Crick genetic duplex having all adenines in one strand and allthymines or uracils in the other strand (Hoogsteen binding; Hoogsteen1959). Subsequently, it was reported that in high salt and at pH valueslower than 7, an all-cytosine polynucleotide, having the cytosinemoieties protonated, can bind in a similar manner to a Watson/Crickduplex having all guanines in one strand and all cytosines in the otherstrand.

Thereafter, is was demonstrated that under high salt and at a pH below7, a polynucleotide containing both cytosines and thymines (or uracils)can bind to a Watson/Crick duplex having the appropriate sequence ofpurines in one strand and pyrimidines in the other strand (Morgan,1968).

In the 1970's this Hoogsteen binding mechanism was exploited foraffinity chromatography purification of duplex genetic fragmentscontaining runs of purines in one strand and pyrimidines in the otherstrand (Flavell, 1975; Zuidema, 1978). In 1987 Dervan and coworkersexploited this Hoogsteen binding mechanism to position an all-pyrimidinepolynucleotide, carrying an EDTA/Fe cleaving moiety, onto a targetgenetic duplex having a specific sequence of purines in one strand andpyrimidines in the other strand (Moser, 1987).

A major-groove binding mode different from the Hoogsteen mode wasreported in the mid-1960's and involves binding of an all-purinepolynucleotide, poly(dI), to a poly(dI)/poly(rC) duplex (Inamn 1964) andto a poly(dI)/poly(dC) duplex (Chamberlin 1965). Similarly, amostly-purine polynucleotide has been recently used by Hogan andcoworkers (Cooney, 1988) for blocking the activity of a selected naturalduplex genetic sequence. These workers reported that in the presence of6 mM Mg⁺⁺ a mostly-purine polynucleotide (24 purines, 3 pyrimidines) ofa specific sequence inhibits transcription of the human C-myc gene in acell-free system.

To date, reported polynucleotides used for binding to genetic duplexesfail to satisfy one or more important criteria for effective use withinliving organisms. Some of these criteria are as follows:

First, the Hoogsteen-binding polynucleotides (polypyrimidines)containing cytosines require a lower-than-physiological pH in order toachieve effective binding (due to the necessity of protonating thecytosine moieties), although it has recently been demonstrated by Dervanand coworkers that the use of 5-methylcytosines in place of cytosinesallows Hoogsteen binding at a pH somewhat closer to physiological(Mahler, 1989), and use of both 5-methylcytosines in place of cytosinesand 5-bromouracils in place of thymines (or uracils) improves bindingstill further (Povsic, 1989).

Second, in the case of polypurine polynucleotides, both inosine(hypoxanthine) and adeninc moieties lack adequate sequence specificityand adequate binding affinity for effective major-groove binding inintracellular applications. The inadequate sequence specificity forinosine (Inman, 1964) and adenine (Cooney, 1988) moieties derives fromthe fact that inosine can bind with similar affinity to the centralpolar major-groove sites of both a C:I (or C:G) base-pair (i.e., NH4 ofC and 06 of G or I) and an A:T or A:U base-pair (i.e., NH6 of A and 04of T or U), and because adenine can bind with similar affinity to thecentral polar major-groove sites of both a T:A or U:A base-pair (i.e.,04 of T or U and NH6 of A) and a G:C base-pair (i.e., 06 of G and NH4 ofC), as discussed further below.

The low binding affinity of inosine for its target base-pairs and ofadenine for its target base-pairs is due to the fact that these purinescan form only two less-than-optimal hydrogen-bonds to the major-groovesites of their respective target base-pairs.

Third, both polypyrimidine and polypurine polynucleotides fail toachieve effective binding to their target genetic duplexes underphysiological conditions, due to the substantial electrostatic repulsionbetween the three closely-packed polyanionic backbones of thethree-stranded complexes. Although this repulsion can be attenuated byhigh salt (Morgan, 1968), divalent cations (Cooney, 1988), or polyamines(Moser, 1987), nonetheless, for applications in living cells, andparticularly cells within intact organisms, control of intracellularcation concentrations is generally not feasible.

Fourth, for therapeutic applications polynucleotides are less thanoptimal because: (i) they are rapidly sequestered by thereticuloendothelial lining of the capillaries, (ii) they do not readilycross biological membranes, and (iii) they are sensitive to degradationby nucleases in the blood and within cells.

For many in vivo applications of sequence-specific duplex-directednucleic acid-binding agents, the principal target is DNA, which appearsto exist within cells predominantly in a B or B-like conformation. Inthis context, polynucleotides which have been used for major-groovebinding to genetic duplexes (Moser, 1987; Cooney, 1988) have a unitbackbone length that is shorter than optimal for binding to duplexgenetic sequences existing in a B-type conformation.

SUMMARY OF THE INVENTION

The present invention includes a polymer composition effective to bindin a sequence-specific manner to a target sequence of a duplexpolynucleotide containing at least two different oriented Watson-Crickbase-pairs at selected positions in the target sequence. The polymer isformed of a specific sequence of subunits selected from the followingforms: ##STR1## where Y is a 2- or 3-atom length, uncharged intersubunitlinkage group; R' is H, OH, or 0-alkyl; the 5'-methylene has a βstereochemical orientation in the 5-membered ring and a uniformstereochemical orientation in the 6-membered ring; R_(i) has astereochemical orientation; and at least about 70% of groups in thepolymer are selected from two or more of the followingbase-pair-specificity groups:

A. for a T:A or U:A oriented base-pair, R_(i) is selected from the groupconsisting of planar bases having the following skeletal ring structuresand hydrogen bonding arrays, where B indicates the polymer backbone:##STR2## where the * ring position may carry a hydrogen-bond donorgroup, such as an amine; and X may be a moiety effective to afforddiscriminative binding on the basis of the moiety at the 5 position ofthe pyrimidine of the target base-pair;

B. for a C:G oriented base-pair, R_(i) is selected from the groupconsisting of planar bases having the following skeletal ring structuresand hydrogen bonding arrays, where B indicates the polymer backbone:##STR3## where the * ring position may carry a hydrogen-bond acceptorgroup, such as a carbonyl oxygen; X may be a moiety effective to afforddiscriminative binding on the basis of the moiety at the 5 position ofthe pyrimidine of the target base-pair; and Z is oxygen or sulfur;

C. for a G:C oriented base-pair, R_(i) is selected from the groupconsisting of planar bases having the following skeletal ring structuresand hydrogen bonding arrays, where B indicates the polymer backbone:##STR4## where the * ring position may carry a hydrogen-bond acceptorgroup, such as a carbonyl oxygen; and X may be a moiety effective toafford discriminative binding on the basis of the moiety at the 5position of the pyrimidine of the target base-pair; and,

D. for an A:T or A:U oriented base-pair, R_(i) is selected from thegroup consisting of planar bases having the following skeletal ringstructures and hydrogen bonding arrays, where B indicates the polymerbackbone: ##STR5## where the * ring position may carry a hydrogen-bonddonating group, such as NH₂ ; and X may be a moiety effective to afforddiscriminative binding on the basis of the moiety at the 5 position ofthe pyrimidine of the target base-pair.

In one embodiment, for use in sequence-specific binding to a duplexnucleic acid sequence in an A conformation, the Y linkage group is twoatoms in length. In another embodiment, for use in sequence-specificbinding to a B-form DNA-DNA duplex nucleic acid sequence, the Y linkagegroup is three atoms in length.

In another embodiment, the invention includes a polymer containingseveral methyl-discriminating bases which afford good target bindingaffinity to a duplex target sequence when said target sequence containsa hydrogen at the 5 position of the pyrimidine of selected base-pairs,but which substantially reduce target binding to a corresponding targetsequence when said target sequence contains a methyl at the 5 positionof the pyrimidine of the selected base-pairs.

In another aspect, the invention includes a method for coupling a firstfree or polymer-terminal subunit having one of the following subunitforms: ##STR6## where R_(i) is a planar ring structure having two ormore hydrogen-bonding sites, with a second free or polymer-terminalsubunit having one of the following subunit forms: ##STR7## where Z is a2-atom or 3-atom long moiety. The method includes i) oxidizing the firstsubunit to generate a dialdehyde intermediate; ii) contacting thedialdehyde intermediate with the second subunit under conditionseffective to couple a primary amine to a dialdehyde; and (iii) adding areducing agent effective to give a coupled structure selected from thefollowing forms: ##STR8##

In still another aspect, the invention provides a method for isolating,from a liquid sample, a target duplex nucleic acid fragment having aselected sequence of base-pairs. The method includes first contactingthe sample with a polymer reagent-containing structure which allowsisolation of the reagent from solution, and attached to this structure,a polymer composition of the type described above, where the polymercomposition has a subunit sequence effective to bind in asequence-specific manner with the selected sequence of base-pairs. Thecontacting is carried out under conditions effective forsequence-specific binding of the polymer composition to the selectedsequence of base-pairs.

Further, the polymers of the present invention can be used to detect thepresence of a target nucleic acid sequence. For example, a support-boundpolymer composition can be contacted with a test solution containing theselected duplex genetic sequence under conditions effective forsequence-specific binding of the polymer composition to its targetsequence of base-pairs. The support-bound polymer with bound selectedduplex genetic sequence is then separated from the test solution. Thepresence of the polymer/target duplex sequence is then detected.Detecting the selected genetic sequence may, for example, utilize one ofthe following: fluorescent compounds, such as, ethidium bromide andpropidium iodide, effective to intercalate into duplex geneticsequences; or reporter moieties linked to oligocationic moietieseffective to bind to the polyanionic backbone of nucleic acids.

Also forming part of the invention is a subunit composition for use informing a polymer composition effective to bind in a sequence specificmanner to a target sequence in a duplex polynucleotide. The compositionincludes one of the following subunit structures: ##STR9## where R' isH, OH, or O-alkyl; the 5'-methylene has a β stereochemical orientationin subunit forms (a), (c), and (d) and a uniform stereochemicalorientation in subunit form (b); X is hydrogen or a protective group ora linking group suitable for joining the subunits in any selected orderinto a linear polymer; Y is a nucleophilic or electrophilic linkinggroup suitable for joining the subunits in any selected order into alinear polymer; and X and Y together are such that when two subunits ofthe subunit set are linked the resulting intersubunit linkage is 2 or 3atoms in length and uncharged; Z is a 2-atom or 3-atom long moiety; and,R_(i), which may be in the protected state and has a β stereochemicalorientation, is selected from the group consisting of planar baseshaving the following skeletal ring structures and hydrogen bondingarrays, where B indicates the aliphatic backbone moiety: ##STR10## wherethe * ring position may carry a hydrogen-bond acceptor group; and X maybe a moiety effective to afford discriminative binding on the basis ofthe moiety at the 5 position of the pyrimidine of the target base-pair;or, where R_(i) is selected from the group consisting of planar baseshaving the following skeletal ring structures and hydrogen bondingarrays, where B indicates the aliphatic backbone moiety: ##STR11## wherethe * ring position may carry a hydrogen-bond donating group; and X maybe a moiety effective to afford discriminative binding on the basis ofthe moiety at the 5 position of the pyrimidine of the target base-pair.

An additional part of the invention is a subunit composition for use informing a polymer composition effective to bind in a sequence specificmanner to a target sequence in a duplex polynucleotide. The compositionincludes one of the following subunit structures: ##STR12## where the5'-methylene has a β stereochemical orientation in subunit forms (b),and (c) and a uniform stereochemical orientation in subunit form (a); Xis hydrogen or a protective group or a linking group suitable forjoining the subunits in any selected order into a linear polymer; Y is anucleophilic or electrophilic linking group suitable for joining thesubunits in any selected order into a linear polymer; and X and Ytogether are such that when two subunits of the subunit set are linkedthe resulting intersubunit linkage is 2 or 3 atoms in length anduncharged; Z is a 2-atom or 3-atom long moiety; and, R_(i), which may bein the protected state and has a β stereochemical orientation, isselected from the group consisting of planar bases having the followingskeletal ring structures and hydrogen bonding arrays, where B indicatesthe aliphatic backbone moiety, the starred atom is not a hydrogen bondacceptor, and W is oxygen or sulfur: ##STR13##

The present invention also includes, a polymer composition capable ofdiscriminating, on the basis of binding affinity, between (a) a selectedtarget sequence in a duplex polynucleotide containing at least onepyrimidine-purine base-pair at a selected position in the targetsequence, where the pyrimidine base has a hydrogen on the 5 ringposition, and (b) the same sequence but in which the pyrimidine of thebase pair at the selected position contains a methyl group on the 5 ringposition of the pyrimidine. The polymer composition includes a polymerhaving the repeating unit ##STR14## where B is a backbone moiety. In thecomposition, at least one R_(i) is selected from the following group:##STR15## where X has a size and shape effective to selectivity reducebinding of R_(i) to a base-pair having a methyl on the 5 ring positionof the pyrimidine.

Another embodiment of the present invention includes a method forinhibiting the biological activity of a selected duplex geneticsequence. In this method, a suitable target sequence of base-pairs isselected within the selected duplex genetic sequence whose activity isto be inhibited. A polymer composition, as described above, is providedwhich is effective to bind in a sequence-specific manner to the targetsequence. The polymer composition is contacted with the selected duplexgenetic sequence under substantially physiological conditions.

This method may further include contacting the polymer composition withthe selected genetic sequence where contacting the polymer compositionwith the selected genetic sequence entails targeting the polymercomposition to a tissue or site containing the selected genetic sequenceto be inactivated. Some methods of delivery include delivering thepolymer composition in the form of an aerosol to the respiratory tractof a patient and/or injecting an aqueous solution of the polymercomposition into a patient.

These and other objects and features of the present invention willbecome more fully apparent when the following detailed description ofthe invention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D illustrate T:A (1A), A:T (1B), C:G (1C) and G:C (1D)oriented Watson-Crick base-pairs, showing the major-groovehydrogen-binding sites of the base-pairs (arrows).

FIGS. 2A and 2B illustrate tautomeric forms of 2-amino pyrimidine (2A),and 2-pyrimidinone (2B), FIGS. 2C and 2D illustrate rigid (2C) andnon-rigid (2D) hydrogen-bonding arrays.

FIG. 3A-3D illustrate standard positioning for a U:A base-pair in an Aconformation and the approximate position of helical axis for an A-formduplex (FIG. 3A), the use of this positioning scheme for assessingR_(a), Θ_(a), and A values for a subunit base hydrogen bonded to thepolar major-groove sites of a U:A base-pair in an A conformation (FIG.3B), the standard positioning for a T:A base-pair in a B conformationand the approximate position of helical axis for a B-form duplex (FIG.3C), and the use of this positioning scheme for assessing R_(b), Θ_(b),and A values for a subunit base hydrogen bonded to the polarmajor-groove sites of a T:A base-pair in a B conformation (FIG. 3D).

FIGS. 4A and 4B illustrates the normal binding (FIG. 4A) andsubstantially reduced binding (FIG. 4B) afforded by a representativemethyl-discriminating base. FIG. 4C illustrates the general skeletalring structure, hydrogen bonding array, and position of themethyl-discriminating moiety of preferred bases designed to bind theirrespective target base-pairs having a hydrogen at the 5 position of thepyrimidine, but not bind their respective target base-pairs when saidbase-pairs have a methyl at the 5 position of the pyrimidine.

FIG. 5A-5C show representative 2'-deoxyribose (FIG. 5A), ribose (FIG.5B), and ribose-derived backbone structures (FIG. 5C) suitable for usein forming the polymer of the invention.

FIGS. 6A-6F show representative morpholino backbone structures suitablefor use in forming the polymer of the invention.

FIGS. 7A-7F show representative acyclic backbone structures suitable forforming the polymer of the invention.

FIG. 8A shows a representative coupled acyclic backbone structure with a4-atom unit backbone length, FIGS. 8B-8C show coupled acyclic backbonestructures with a 5-atom unit backbone length, and FIGS. 8D-8F showcoupled acyclic backbone structures with a 6-atom unit backbone length.

FIGS. 9A-9D show representative coupled cyclic backbone structures witha 6-atom unit backbone length, and FIGS. 9E-9F show representativecoupled cyclic backbone structures with a 7-atom unit backbone length.

FIGS. 10A and 10B illustrate a guanine base and its binding to a C:Goriented Watson-Crick base-pair (FIG. 10A) and a diaminopurine base andits binding to a T:A oriented Watson-Crick base-pair (FIG. 10B). FIG.10C shows tetramer formation with guanine bases. FIG. 10D shows hydrogenbonding at diaminopurine bases. FIG. 10E shows several guanine analogsthat lack a hydrogen bond acceptor at the 7 position.

FIG. 11A and 11B show hydrogen bonding of a cytosine base to a G:C (FIG.11A) and T:A (FIG. 11B) oriented base-pair.

FIGS. 12A and 12B show hydrogen bonding of a uracil base to an A:T (FIG.12A) and C:G (FIG. 12B) oriented base-pair.

FIGS. 13A-13D illustrate the general skeletal ring structure, hydrogenbonding array, and backbone attachment position of a tautomeric basedesigned for binding to a G:C or A:T Watson-Crick base-pair (FIG. 13A),and three specific embodiments of the 13A structure (FIGS. 13B-13D).

FIGS. 14A and 14B show the hydrogen bonding of the FIG. 13B structure toa G:C (FIG. 14A) and A:T (FIG. 14B) oriented base-pair.

FIG. 15A-15D illustrate the general skeletal ring-structure, hydrogenbonding array, and backbone attachment position of a base designed forbinding to a G:C Watson-Crick base-pair (FIG. 15A), and three specificembodiments of the FIG. 15A structure (FIGS. 15B-15D).

FIG. 16 shows the hydrogen bonding of the FIG. 15D structure to a G:Coriented base-pair.

FIG. 17A illustrates the general skeletal ring-structure, hydrogenbonding array, and backbone attachment position of a base designed forbinding to a G:C Watson-Crick base-pair, and FIG. 17C shows a specificembodiment of the FIG. 17A structure hydrogen bonded to a G:C orientedbase-pair; FIG. 17B illustrates the general skeletal ring-structure,hydrogen bonding array, and backbone attachment position of a relatedbase designed for binding to an A:T or A:U Watson-Crick base-pair, andFIG. 17D shows a specific embodiment of the 17B structure hydrogenbonded to a A:T oriented base-pair.

FIGS. 18A-18D illustrate the general skeletal ring-structure, hydrogenbonding array, and backbone attachment position of a base designed forbinding to a G:C Watson-Crick base-pair (FIG. 18A), and three specificembodiments of the 18A structure (FIGS. 18B-18D).

FIG. 19 shows the hydrogen bonding of the FIG. 18B structure to a G:Coriented base-pair.

FIG. 20A-20D illustrate the general skeletal ring-structure, hydrogenbonding array, and backbone attachment position of a base designed forbinding to an A:T or A:U Watson-Crick base-pair (FIG. 20A), and threespecific embodiments of the 20A structure (FIGS. 20B-20D).

FIG. 21 shows the hydrogen bonding of the FIG. 20D structure to an A:Toriented base-pair.

FIGS. 22A-22D illustrate the general skeletal ring-structure, hydrogenbonding array, and backbone attachment position of a base designed forbinding to an A:T or A:U Watson-Crick base-pair (FIG. 22A), and threespecific embodiments of the 22A structure (FIGS. 22B-22D).

FIG. 23 shows the hydrogen bonding of the FIG. 22B structure to an A:Toriented base-pair;

FIG. 24A illustrates the general skeletal ring-structure, hydrogenbonding array, and backbone attachment position of a base designed forbinding to an C:G Watson-Crick base-pair, and FIG. 24C illustrates aspecific embodiment of the FIG. 24A structure; FIG. 24B illustrates thegeneral skeletal ring-structure, hydrogen bonding array, and backboneattachment position of a related base also designed for binding to anC:G Watson-Crick base-pair, and FIG. 24D illustrates a specificembodiment of the FIG. 24B structure.

FIG. 25 shows the hydrogen bonding of the FIG. 22C structure to a C:Goriented base-pair.

FIG. 26A illustrates the general skeletal ring-structure, hydrogenbonding array, and backbone attachment position of a base designed forbinding to a T:A or U:A Watson-Crick base-pair, and FIGS. 26C-26Dillustrate two specific embodiments of the FIG. 26A structure; FIG. 26Billustrates the general skeletal ring-structure, hydrogen bonding array,and backbone attachment position of a related base also designed forbinding to a T:A or U:A Watson-Crick base-pair, and FIG. 26E illustratesa specific embodiment of the FIG. 26B structure.

FIG. 27 shows the hydrogen bonding of the FIG. 26C structure to a T:Aoriented base-pair.

FIG. 28A illustrates the general skeletal ring-structure, hydrogenbonding array, and backbone attachment position of a base designed forbinding to a T:A or U:A Watson-Crick base-pair, and FIG. 28B illustratesa specific embodiment of the FIG. 28A structure.

FIG. 29A illustrates the general skeletal ring-structure, hydrogenbonding array, and backbone attachment position of a base designed forbinding to a C:G Watson-Crick base-pair, and FIG. 29B illustrates aspecific embodiment of the 29A structure.

FIG. 30A illustrates the general skeletal ring-structure, hydrogenbonding array, and backbone attachment position of a base designed forbinding to an A:T or A:U Watson-Crick base-pair, and FIGS. 30C-30Eillustrate three specific embodiments of the FIG. 30A structure; FIG.30B illustrates the general skeletal ring-structure, hydrogen bondingarray, and backbone attachment position of a related base also designedfor binding to an A:T or A:U Watson-Crick base-pair, and FIG. 30Fillustrates a specific embodiment of the FIG. 30B structure.

FIG. 31A illustrates the general skeletal ring-structure, hydrogenbonding array, and backbone attachment position of a base designed forbinding to a G:C Watson-Crick base-pair, and FIG. 31C illustrates aspecific embodiment of the FIG. 31A structure; FIG. 31B illustrates thegeneral skeletal ring-structure, hydrogen bonding array, and backboneattachment position of a related base also designed for binding to a G:CWatson-Crick base-pair, and FIG. 31D illustrates a specific embodimentof the FIG. 31B structure.

FIG. 32A shows the hydrogen bonding of the FIG. 28B structure to a U:Aoriented base-pair; FIG. 32B shows the hydrogen bonding of the FIG. 29Bstructure to a C:G oriented base-pair; FIG. 32C shows the hydrogenbonding of the FIG. 30D structure to an A:T oriented base-pair; FIG. 32Dshows the hydrogen bonding of the FIG. 31C structure to a G:C orientedbase-pair.

FIG. 33 illustrates the coupling cycle used in an exemplary solid-phasesynthesis of one embodiment of the binding polymer of the presentinvention.

FIG. 34, 35 and 36 illustrate segments of three polymers constructedaccording to the invention, with each designed to bind to a region of anA-form genetic duplex nucleic acid having the sequence of base-pairs:C:G, A:T, T:A, and G:C.

FIG. 37 illustrates a segment of a polymer constructed according to theinvention, and designed to bind to a region of an A-form RNA/RNA duplexhaving the sequence of base-pairs: C:G, A:U, U:A, and G:C.

FIG. 38 illustrates a segment of a polymer constructed according to theinvention, and designed to bind to a segment of duplex DNA in the Bconformation in the transcriptional control region of atranscriptionally-active gene, with that duplex target sequence havingthe sequence of base-pairs: T:A, G:5mC, 5mC:G, and A:T.

FIG. 39 illustrates the coupling cycle in a novel method for assemblingnucleic acid-binding polymers.

DETAILED DESCRIPTION OF THE INVENTION

I. Polymer Subunit Construction

The polymer of the invention is designed for binding with base-pairspecificity to a selected sequence (the target sequence) in a strand ofduplex nucleic acid. As used herein, duplex sequence refers to asequence of contiguous oriented Watson/Crick base-pairs, where the fouroriented base-pairs are: A:T (or A:U), T:A (or U:A), G:C, and C:G, whereA, T, U, G, and C, refer to adenine, thymine, uracil, guanine, andcytosine nucleic acid bases, respectively.

The polymer is formed of subunits, each of which comprises a backbonestructure and linkage group, which collectively form an unchargedbackbone, and a base attached to the backbone structure, which providesbase-pair-specific hydrogen-bonding to the target. The requirements ofthe backbone structure, linkage group, and attached base in the polymersubunits are described in detail below.

In the context of the duplex binding polymers of the present invention,the term "base" refers to planar base-pair-specific hydrogen-bondingmoieties.

A. Subunit Base Requirements

Because of the symmetry of the polar minor-groove sites and theasymmetry of polar major-groove sites in Watson/Crick base-pairs, toachieve a given level of sequence specificity a minor-groove-bindingagent would have to recognize twice as many base-pairs as would acorresponding major-groove-binding agent. Accordingly, hydrogen-bondingof the subunit base is to the polar sites in the major groove of thetarget duplex.

FIGS. 1A-1D shows T:A, A:T, C:G, and G:C oriented Watson/Crickbase-pairs, with the major-groove hydrogen-bonding sites indicated byarrows in the figure. For the T:A and A:T oriented base-pairs, the polarmajor-groove sites include the N7 and a hydrogen on the N6 of adenineand the 04 of thymine (or uracil). For the C:G and G:C orientedbase-pairs, the polar major-groove sites include the 06 and N7 ofguanine and a hydrogen on the N4 of cytosine.

In order to make a significant contribution to the free energy ofbinding and to provide adequate base-pair specificity, the subunit baseshould form at least two hydrogen bonds to its target base-pair. Thatis, each subunit base in the polymer should contain at physiological pHa hydrogen-bonding array suitable for binding to two or three of thepolar major-groove sites on its respective oriented target base-pair.Table 1 shows the hydrogen-bonding arrays comprising the polarmajor-groove sites for each of the four oriented Watson/Crickbase-pairs, and the corresponding hydrogen-bonding array of the subunitbase suitable for hydrogen-bonding to said polar major-groove sites.

In the table, X is generally an N, O, or S atom, but can also be F, Cl,or Br, having a non-bonded pair of electrons suitable for hydrogenbonding, and ** represents the nonbonded pair of electrons suitable forhydrogen-bonding.

                  TABLE 1                                                         ______________________________________                                        Oriented                                                                              hydrogen-bonding                                                                            Required hydrogen-bonding                               base-pair                                                                             array of base-pair                                                                          array of subunit base                                   ______________________________________                                        A:T     **     H       **   N    X   or   N   X   N                                                       H    **       H   **  H                           T:A     **     H       **   X    N   or   N   X   N                                                       **   H        H   **  H                           C:G     H      **      **   N    N   or   X   N   N                                                       H    H        **  H   H                           G:C     **     **      H    N    N   or   N   N   X                                                       H    H        H   H   **                          ______________________________________                                    

As indicated above, the polymer subunit base typically contains thespecified hydrogen-bonding array at physiological pH (in contrast tocytosine moieties used for Hoogsteen-type major-groove binding). Thisassures that at physiological pH, binding of the subunit base makes asubstantial contribution to the free energy of binding between thepolymer and its target duplex.

At physiological pH the subunit base should be predominantlynon-ionized. More specifically, basic moieties should have pKb values ofat least 7.5 or greater, and acidic moieties should have pKa values ofat least 7.7 or greater. This lack of substantial ionic charge providestwo advantages. First, for applications in living cells, the lack ofionic groups on the binding polymers facilitates passage of the polymeracross biological membranes. Second, lack of negative charges avoids theproblem of charge repulsion between the binding polymer and thenegatively charged phosphates of its target duplex.

Major-groove hydrogen-bonding arrays of the four oriented Watson/Crickbase-pairs are illustrated in Table 2.

In Table 2, H is a hydrogen bound to a nitrogen, and ** is an electronpair of nitrogen or oxygen available for hydrogen bonding.

                  TABLE 2                                                         ______________________________________                                                      NH4    O6  N7          N7  O6  NH4                              ______________________________________                                        C:G           H      **  **  G:C      ** **  H                                A:T(U)  **    H      **      T(U):A      **  H    **                                  N7    NH6    O4                  O4  NH6  N7                          ______________________________________                                    

The respective positioning of the base-pair H-bonding arrays shown inTable 2, which approximates their relative positions in the major-grooveof a duplex genetic sequence, illustrates the fact that two of theH-bonding sites of a C:G base-pair (NH4 and 06) are positioned nearlythe same as two of the H-bonding sites of an A:T(U) base-pair (NH6 and04). Likewise, two of the hydrogen-bonding sites of a G:C base-pair (06and NH4) are positioned nearly the same as two of the H-bonding sites ofa T(U):A base-pair (04 and NH6). Because of these similarities inpositioning between central hydrogen-bonding sites of the orientedbase-pairs, subunit bases which hydrogen-bond only to the polar sitesnear the center of the major-groove (underlined in the above table) lackadequate specificity for a given base-pair. Accordingly, in order for asubunit base to achieve high specificity for a single orientedbase-pair, the base should hydrogen bond to the N7 of its respectivetarget base-pair.

If a subunit base is targeted to bind only one of the four orientedWatson/Crick base-pairs, the tautomeric state of that subunit baseshould be sufficiently fixed under conditions of use so that at leasttwo of the hydrogen-bonding groups positioned for base-pair binding willnot tautomerize to give a structure capable of H-bonding with comparableaffinity to a base-pair other than the targeted base-pair To illustrate,FIG. 2A shows an acceptably fixed structure (2-amino pyrimidine, whichexists almost exclusively in the 2-amino tautomeric form). FIG. 2B showsa second structure which lacks specificity for a single base-pair due toits facile tautomerization under physiological conditions(2-pyrimidinone). Dominant tautomeric forms of a wide assortment ofrepresentative heterocyclic structures have been tabulated in a bookedited by Elguero, Marzin, Katritzky & Linda (1976).

The subunit bases typically have structures which provide a relativelyrigid arrangement of at least two of the base-pair H-bonding groupspositioned for base-pair binding. Such rigidity is best afforded by aring structure wherein at least two of the polar hetero atoms to beinvolved in H-bonding to the target base-pair are either part of thering or directly attached to the ring. To illustrate, FIG. 2C shows astructure (2-amino-3-cyano pyrrole) which satisfies this rigidityrequirement. FIG. 2D shows a structure (2-carboxamide pyrrole) whichfails to satisfy the requirement.

The simplest sequence-specific binding polymers are those which bind toa target which is composed of contiguous base-pairs in thepolynucleotide duplex. This, in turn, requires that the subunit bases ofthe binding polymer be no thicker than the target base-pairs to whichthey are to bind. Accordingly, each subunit structure should be planar.This is best achieved by using subunit bases having aromatic characterand/or having plane trigonal bonding for most or all ring atoms.

B. Constraints on the Position and Angle of Attachment of the Base tothe Backbone

Considering now the geometric requirements of the polymer subunits, mostduplex nucleic acids adopt either of two general conformations. RNA/RNAand RNA/DNA duplexes adopt an A-type conformation. DNA/DNA duplexesadopt a B-type conformation, but can readily convert to an Aconformation under certain conditions, such as high salt or low polaritysolvent.

In duplex nucleic acids the polar major-groove sites on each of theWatson/Crick base-pairs are fairly regularly positioned with respect tocorresponding arrays of major-groove sites on neighboring base-pairs,with the relative positions being defined by the helical conformationparameters of axial position, axial rise, and axial rotation.

In principle, the backbone attachment positions of the different subunitbases, when the bases are hydrogen-bonded to their respective targetbase-pairs, need not be positioned in any regular way relative to theirtarget base-pairs. However, when there is significant variability in therelative backbone attachment positions of the different subunit basesrelative to their target base-pairs, each of the backbone structures ofthe component subunits in the polymer are custom tailored with respectto backbone length and position of subunit base attachment, leading toextremely high development and production costs.

However, if all of the subunit bases of a given subunit set have similarbackbone attachment positions and angles relative to their respectivetarget base-pairs, then all subunits of the set can have identicalbackbone structures, greatly simplifying the synthetic effort requiredfor polymer construction. To this end, the polymer subunits used in thepresent invention are selected, according to criteria described below,to have similar backbone attachment positions and angles.

To understand what is meant by similar backbone attachment positions andangles, reference is made to FIG. 3A, which shows a Watson/Crickbase-pair (W/C bp) positioned relative to the helical axis (denotedH_(a)) of an A-form genetic duplex, i.e., (A, 12, 0.326) RNA. The lowerhorizontal line in the figure connects the two ribose C1' atoms of theWatson-Crick base-pair, and the vertical line (denoted PB) is theperpendicular bisector of the first-mentioned line.

The backbone attachment position and angles of a subunit base are thendetermined by positioning the subunit base on its corresponding targetbase-pair in this standardized position, with the subunit base beinghydrogen bonded to the appropriate polar major-groove sites on theWatson-Crick base-pair, as shown for a 2,6-diaminotriazine subunit basein FIG. 3B.

The backbone attachment position of the subunit base, relative to itsA-form target duplex, can then be described by an R_(a) and Θ_(a) value,where R_(a), is the radial distance, in angstroms, from the helical axisof the A-form target duplex to the center of the backbone atom (denotedB; FIG. 3B) to which the subunit base is attached, and Θ_(a) is theangle, in degrees, about this helical axis, measured clockwise from theperpendicular bisector to the center of the aforementioned backboneatom. The attachment angle, A, is defined as the angle, in degrees,measured clockwise from the perpendicular bisector, between theperpendicular bisector and a line parallel to the bond between thesubunit base and the backbone moiety.

FIG. 3B illustrates R_(a), Θ_(a), and A parameters for a2,6-diaminotriazine subunit base hydrogen-bonded to a U:A base-pair inan A conformation. FIG. 3C illustrates a correspondingly positionedbase-pair of a B-form duplex, and FIG. 3D illustrates R_(b), Θ_(b), andA parameters for this 2,6-diaminotriazine subunit base hydrogen-bondedto a T:A base-pair in a B conformation.

In order to unambiguously define the target base-pair for a selectedsubunit base with a given backbone attachment site, two orientations foreach Watson/Crick base-pair in the target duplex must be considered. Theresultant 4 oriented base-pairs are designated as A:T, T:A, C:G, and G:C(and corresponding base-pairs where U replaces T). The orientations ofthese base-pairs are defined in Table 3.

                  TABLE 3                                                         ______________________________________                                        Oriented Base-pair                                                                           Θ value for N7 of Purine                                 Designation    of Target Base-pair                                            ______________________________________                                        A:T (A;U)      >180°                                                   T:A (U:A)      <180°                                                   C:G            <180°                                                   G:C            >180°                                                   ______________________________________                                    

In principle, the backbone attachment position for any given subunitbase, in position on its target base-pair, can have a Θ value, , X°, inthe range of 0° to 180°. By flipping the target base-pair, the Θ valueof that same target-bound subunit base is changed to 360°-X°. Theconvention used in the following discussion is that the Θ value for eachsubunit base of the binding polymer is less than 180°.

Thus, in the context of selecting a subunit set suitable for assemblingthe binding polymers disclosed herein, to explicitly define whichorientation of a given base-pair constitutes the target for a specifiedsubunit base, it is important to designate the orientation of thattarget base-pair such that the backbone attachment position of thebase-pair-bound subunit base has a Θ value less than 180°. Toillustrate, a 2,6-diaminotriazine subunit base having a backbone moietyattached through the C4 of the triazine (FIG. 3B) can bind to a U:Abase-pair in an A conformation to give a Θ value of 28°. When this samesubunit base is hydrogen-bonded to that same base-pair in thebase-pair's opposite orientation (ie., A:U), the Θ value for the subunitbase is 332° (ie., 360°-28°). The convention used herein dictates thatthe target base-pair for this subunit base is U:A (where Θ is <180°),and not A:U (where Θ is >180°).

Acceptable values of R, Θ, and A for prospective recognition moietiescan be readily obtained with CPK molecular models (The Ealing Corp.,South Natick, Mass., USA). More accurate values can be estimated byoptimization of the hydrogen-bonding in the subunit base/base-pairtriplex via a computer molecular mechanics program, such as areavailable commercially. The subunit bases should be so selected that agiven subunit set (the set of subunits used in assembly of a givenpolymer) all have R values within about 2 angstroms of each other, Θvalues within about 20° of each other, and A values within about 30° ofeach other.

In order for a subunit base to have a high specificity for only one ofthe oriented base-pairs, it is important that the subunit base not beable to bind to a given base-pair in both orientations (e.g., G:C andC:G) simply by rotation of the subunit base about its linkage to itsbackbone structure. Therefore, the earlier-described backbone attachmentposition or angle should be asymmetrical with respect to the C1'positions of the target base-pair. Specifically, Θ_(a) for the subunitbase should have a value greater than about 10°, or the attachmentangle, A, for the subunit base should have a value greater than about25°.

C. Bases Suitable for Binding Discrimination on the Basis of the Moietyat the 5 Position of the Pyrimidine of the Target Base-Pair

Subunits have been devised which can be used to prepare a polymer whichis capable of binding its duplex target genetic sequence when saidsequence contains a hydrogen at the 5 position of the pyrimidine ofseveral specified base-pairs, but which is not capable of binding acorresponding duplex target genetic sequence when said sequence containsa methyl at the 5 position of the pyrimidine of said specifiedbase-pairs.

One application of polymers having this methyl-discriminating propertyis the selective inactivation of duplex RNA sequences (e.g., thereplicative intermediate of an RNA virus) without concomitantinactivation of a corresponding duplex DNA sequence (e.g., an identicalsequence in a patient's inherent cellular DNA). Another application ofsuch a methyl-discriminating polymer is for selective attack on atranscriptionally-active oncogene (containing clusters of CpG sequencesin the transcriptional control region) in cancer cells, withoutconcomitant attack on the corresponding transcriptionally-inactiveproto-oncogene (containing clusters of 5mC:G sequences in thetranscriptional control region) in normal cells.

The essential characteristic of these methyl-discriminating bases isthat they contain a special discriminating moiety which is of the propersize and shape and is so positioned that when the base is hydrogenbonded to its target base-pair the discriminating moiety occupies aportion of that space which would be occupied by a methyl at the 5position of the pyrimidine of the target base-pair, but does not occupythat space which is occupied by a hydrogen at the 5 position of thepyrimidine of the target base-pair. As a consequence, such amethyl-discriminating base exhibits normal binding affinity for itsnon-methylated target base-pair, but because of steric exclusion itexhibits substantially reduced binding affinity for its correspondingtarget base-pair containing a methyl at the 5 position of thepyrimidine.

FIG. 4 illustrates such methyl-discriminatory binding. FIG. 4A shows thenormal binding of such a base to its non-methylated target base-pair(C:G), and FIG. 4B shows the substantial disruption of binding of thesame base to its corresponding methylated target base-pair (5mC:G). FIG.4C illustrates the general skeletal ring structure, hydrogen bondingarray, backbone attachment position, and position of themethyl-discriminating moiety, R_(d), of preferred bases designed to bindtheir respective target base-pairs having a hydrogen at the 5 positionof the pyrimidine, and designed not to bind their respective targetbase-pairs when said base-pairs have a methyl at the 5 position of thepyrimidine.

In regard to the 5-6-5 fused-ring bases (Structures i through vi of FIG.4C), in order to effectively restrict binding to their respectivemethylated target base-pairs, the R_(d) moiety should be larger than ahydrogen, oxygen, or fluorine. When R_(d) in these bases is a methyl itaffords only a modest reduction in binding affinity of the bases fortheir methylated target base-pairs. Larger moieties, such as sulfur,chlorine, bromine, cyano, azido, and similar sized moieties, provide agreater disruption of binding to a methylated target base-pair, whilestill affording effective binding to the corresponding non-methylatedbase-pair. Even better binding discrimination is afforded bydifluoromethyl and trifluoromethyl moieties, which still affordeffective binding to the corresponding non-methylated base-pairs. Inthis regard, the difluoromethyl moiety is preferred when the backbonelinkage is on the ring containing the R_(d) moiety, as in structures iiiand vi of FIG. 4C, and the trifluoromethyl moiety is preferred when thebackbone linkage is not on the ring containing the R_(d) moiety, as instructures i, ii, iv, and v of FIG. 4C.

While still larger R_(d) moieties, such as isopropyl, t-butyl,dichloromethyl, and trichloromethyl, can also affordmethyl-discriminatory binding, nevertheless, they are undesirablebecause they also interfer with effective stacking of contiguous basesof the polymer when said polymer is in position on its duplex targetsequence.

In regard to the 6-5-5 fused-ring bases (Structures vii and viii of FIG.4C), in order to effectively restrict binding to their respectivemethylated base-pairs, the R_(d) moiety should be larger than ahydrogen. When the methyl-discriminating moiety is oxygen, fluorine, ora methyl it affords substantial reduction of binding to the methylatedtarget base-pair, while still affording good binding affinity to thenon-methylated target base-pair. Larger R_(d) moieties on the 6-5-5 typebases are undesirable because they tend to compromise binding to thenon-methylated target base-pairs.

Other R_(d) moieties can be selected to afford methyl-discriminatorybinding for the above described bases. Selection of these moieties canbe based on the guidance presented above and other structure-functionrelationship analyses (e.g., Dehlinger, 1992).

D. Backbone Structure Constraints

This section considers the backbone structure constraints for a selectedsubunit set. Principally, the subunit should be joinable in any selectedorder to other subunit structures via uncharged linkages having thegeneral properties discussed in Section E below ("IntersubunitLinkages"). Further, the subunit backbone structures and linkages mustprovide proper spacing and allow correct orientation and positioning oftheir respective subunit bases for effective binding of the subunitbases to their respective oriented base-pairs in the target duplexsequence.

A principal requirement for the subunit backbone structure and linkageis that it provide a means for joining the subunits in essentially anyspecified order. This requirement can be satisfied by structurescontaining either heterologous or homologous linking groups.Heterologous type backbone moieties contain a nucleophilic group (N) onone end and an electrophilic group (E) on the other end, as illustratedbelow.

    N--E

The preferred functional groups for the N component include primary andsecondary amine, hydrazine, hydroxyl, sulfhydryl, and hydroxylamine. Thepreferred functional groups for the E component include the followingacids and derivatives thereof: carboxylic, thiocarboxylic, phosphoric,thiophosphoric, esters, thioesters, and amides of phosphoric andthiophosphoric, phosphonic and thiophosphonic, and sulfonic acid. Othersuitable E groups include aldehyde, dialdehyde (or vicinal hydroxylssuitable for conversion to a dialdehyde), alkyl halide, and alkyltosylate.

Homologous type backbone moieties can be of two types, one type havingnucleophilic end groups and the other type having electrophilic endgroups; or, a single homologous backbone moiety can be alternated withan appropriate linker. These alternatives are illustrated below:

    N--N alternated with E--E

    N--N alternated with E linker

    N linker alternated with E--E

Preferred functional groups for N and E are as in the heterologousbackbone moieties. Preferred E linkers include carbonyl; thiocarbonyl;alkyl, ester, thioester, and amide of phosphoryl and thiophosphoryl;phosphonyl and thiophosphonyl; sulfonyl; and, oxalic acid. A preferred Nlinker is 1,2-Dimethylhydrazine.

The present invention contemplates a variety of both cyclic and acyclicbackbone structures, as will be illustrated in FIGS. 5-9 below. Onelimitation of acyclic backbone structures is that activation of theelectrophilic linking groups preparatory to polymer assembly, can leadto varying amounts of undesired intramolecular attack on sites of thesubunit base. By contrast, with properly structured cyclic backbonemoieties, the activated electrophile can be effectively isolated fromreactive sites on the subunit base, thereby reducing unwantedintramolecular reactions.

However, use of aliphatic cyclic backbone moieties does entail thepresence of multiple chiral centers in each backbone structure. Withproper selection of cyclic backbone structures, synthetic challengesassociated with such multiple chiral centers can be largelycircumvented, by utilizing readily available natural products for thebackbone moiety or, preferably, for the entire subunit, or as a proximalprecursor thereto.

This preference for backbone structures, or entire subunits, fromnatural sources reflects the difficulty, and corresponding greaterexpense, of de novo preparation of aliphatic ring structures havingmultiple chiral centers. Accordingly, preferred categories of cyclicbackbone moieties are those comprising, or readily derived from,deoxyribose or ribose. In addition, certain other natural cyclicstructures wherein a single enantiomer is available, or can be readilyprepared or isolated, are also preferred. FIGS. 5A-5C illustrateexemplary cyclic backbone structures comprising or derived fromdeoxyribosides or ribosides. R' in FIG. 5 indicates H or alkyl, andR_(i) indicates the subunit base, which, as seen, has the sameβ-orientation as natural nucleosides. FIGS. 6A-6F illustrate exemplarycyclic morpholino backbone structures derivable from ribosides, havingeither a β-orientation (FIGS. 6A-6C) or an α-orientation (FIGS. 6D-6F)for the 5'-methylene (numbered as in the parent ribose), again with a βorientation of the R_(i) base. The synthesis of such subunits will bedescribed below and in Examples 1-5. FIGS. 7A-7F show representativetypes of acyclic backbone structures.

E. Intersubunit Linkages

This section considers several types and properties of intersubunitlinkages used in linking subunits to form the polymer of the invention.First, the backbone is stable in neutral aqueous conditions. Since thebinding polymers are designed for use under physiological conditions itis necessary that the intersubunit linkages be stable under saidconditions. The linkages are also stable under those conditions requiredfor polymer assembly, deprotection, and purification. To illustrate thisstability requirement, an alkyl sulfonate (R--(SO₂)--O--CH₂ --R') isprecluded because the resultant structure is unduly sensitive tonucleophilic attack on the CH₂. Further, while carbonates(R--O--(C═O)--O--R') and esters (R--(C═O)--O--R') can be successfullyprepared, their instability under physiological conditions renders themof little practical value.

Secondly, the backbone is adaptable to a conformation suitable fortarget binding. If the intersubunit linkage is such that it exhibitsspecific rotational conformations (as is the case for amides,thioamides, ureas, thioureas, carbamates, thiocarbamates, carbazates,hydrazides, thiohydrazides, sulfonamides, sulfamides, andsulfonylhydrazides) then it is important either that the rotomercompatible with target binding be the lowest energy conformation, orthat the barrier to rotation between the conformations be relatively low(ie., that the conformations be rapidly interchangeable at physiologicaltemperatures). Thus, a secondary amide (N-alkyl amide, which prefers toadopt a trans conformation) would be acceptable if the transconformation is suitable for pairing to the target duplex. By contrast,tertiary amides and related N,N-dialkyl structures generally have twoapproximately equal low energy conformations, and so to be useful in abinding polymer, the linkages should have a relatively low energybarrier to interconversion between the two conformations.

The barrier to rotation between two conformers can be assessed by NMRessentially as follows: At a temperature where the two conformers areinterconverting slowly relative to the NMR time scale (on the order of10⁻ 8 sec) two distinct signals are often seen, each representing asingle conformer. As the NMR spectra are taken at progressively highertemperatures, the two conformer signals coalesce--indicating rapidinterconversion. Thus, the coalescence temperature (Tc) provides auseful measure of the rotational freedom of various linkage types. Forexample, N,N-dimethylformamide exhibits a Tc of about 114° C.(Bassindale, 1984) and conformers of analogous tertiary amides have beenfound to interconvert slowly in biological macromolecules. By contrast,experiments performed in support of the present invention demonstratethat an N,N-dialkyl carbamate-containing structure exhibits a Tc justunder 44° C. indicating reasonable conformational freedom atphysiological temperature.

An N,N-dialkylsulfinamide (which should have a rotational energy barriersimilar to that of sulfonamide and related substances) has been reportedto have a Tc lower than minus 60° C. (Tet. Let. 10 509 (1964)). Based onthese considerations, backbone linkages containing N,N-dialkyl-typecarbamate, thiocarbamate, carbazate, and various amidates of phosphorousand sulfur are preferred, while N,N-dialkyl-type amide, thioamide, urea,thiourea, hydrazide, and thiohydrazide linkages are generallyunacceptable.

Third, the backbone should be uncharged. For therapeutic applications itis desirable to design these binding polymers so that they i) are notsequestered by the reticuloendothelial lining of the capillaries; ii)readily cross cell membranes; iii) are resistant to degradation bynucleases; and, iv) are not repelled by the high density of negativecharge on the backbones of the target duplex. These design objectivesare best achieved by using both intersubunit linkages and backbonemoieties which are largely uncharged (non-ionic) at physiological pH.

When the subunit bases are positioned on contiguous base-pairs of theirtarget sequence via hydrogen-bonding, and if all recognition moieties ofthe subunit set have well matched R, Θ, and A values, then the distancefrom the subunit base attachment position of one backbone moiety to theattachment position of the next backbone moiety is the square root ofthe following formula:

    (R sine(rot)).sup.2 +(R cosine(rot)-R).sup.2 +(rise).sup.2

where R is the distance from the helical axis to the center of the atomof the backbone moiety to which the subunit base is attached, rot is theaxial rotation value for the target duplex (typically about 30° to 33°for an A-form duplex and 36° for a B-form duplex), and rise is the axialrise value for the target duplex (typically about 2.8 to 3.3 A for anA-form duplex and 3.4 A for a B-form duplex). It is this distance whichmust be spanned by the unit backbone length of the binding polymer,i.e., the length of one backbone structure plus the intersubunit linkagebetween backbone structures. However, A-form (RNA/RNA and RNA/DNAduplexes) and B-form (DNA/DNA) target duplexes are both somewhatflexible. Accordingly, these duplexes can generally accommodate bindingpolymers which have unit backbone lengths that are a fraction of anangstrom shorter or longer than the calculated length requirement.Further, DNA/DNA in a B conformation can be converted to an Aconformation under certain conditions.

In selecting a particular backbone structure, the following factors bearon the required length and so should be taken into consideration: first,any conformational restrictions imposed by hindered rotations aboutbonds such as amides and carbamates; second, when the subunit bases arein position on their target base-pairs, any steric interactions betweenthese bases and the target duplex, and between the bases and the polymerbackbone; third, steric interactions between different components of thebackbone structure; and fourth, for cyclic backbone moieties, favoredconformations of the component ring structure of the subunit backbonestructures.

One way to determine whether or not a prospective polymer backbone islikely to be acceptable for use against a particular target conformation(e.g., A-form or B-form) is to assemble, with CPK molecular models, arepresentative target genetic duplex in the desired conformation, withsubunit bases H-bonded thereto. The prospective polymer backbone is thenadded to the model. Criteria for the evaluation of backbone operabilityas a duplex binding polymer include the following: (i) the prospectivepolymer backbone can be easily attached without having to adopt anenergetically unfavorable conformation, and (ii) the attachment of thepolymer backbone does not cause significant perturbation of the targetstructure, and if there are no unacceptable steric interactions.Additional support for the suitability of a prospective backbonestructure can be obtained by modeling the polymer/target triplex on acomputer using a molecular mechanics program to obtain an optimizedbonding structure via an energy minimization procedure. Such modelingcan help to identify significant unfavorable interactions (e.g.,dipole-dipole repulsions) that might be overlooked in the initial CPKmodeling.

As noted above, such factors as R, Θ, and A values for the subunit basesof a given subunit set, and steric and rotational constraints ofparticular subunit structures and intersubunit linkages, bear on howlong a unit backbone must be in order to provide the correct spacing ofsubunit bases for binding to a target duplex in a given conformation.However, as a rule, subunit sets wherein the subunit bases of the sethave R_(a) values less than about 7 angstroms and Θ values clusteredwithin about 15° of each other, and A values clustered within about 30°of each other, generally require a 4-atom or 5-atom unit-lengthacyclic-type backbone, such as shown in FIGS. 8A-8C, or a 6-atomunit-length cyclic-type backbone, such as shown in FIG. 9A-9D, forbinding to target duplexes in an A-type conformation.

Subunit base sets having Rb values less than about 11 Angstroms, Θ_(b)values within about 15° of each other, and A values clustered withinabout 30° of each other generally require a 6-atom unit-lengthacyclic-type backbone, such as shown in FIGS. 8D-8F, or a 7-atomunit-length cyclic-type backbone, such as shown in FIGS. 9E-9F, forbinding to target duplexes in a B-type conformation.

However, it should be noted that DNA/DNA duplexes, which generally existin a B conformation, can readily convert to an A conformation. Two suchconditions which cause this B to A transition are high salt and lowpolarity solvent. It also appears that a B to A conformationaltransition of the target duplex can be induced by duplex-directedbinding polymers having backbone unit-lengths shorter than optimal forbinding to a B-form duplex. However, such conformation transitions incura cost in free energy of binding, and so, to compensate, the bindingpolymer's affinity for its target is increased accordingly. Because ofthe feasibility of this B to A conformational transition of targetduplexes, for some applications the shorter unit-length backbonessuitable for A-form target duplexes can also be used for targetinggenetic sequences which exist normally in a B conformation.

F. Subunit Sets

Subunits of a set can be assembled in any desired order for targeting aselected duplex sequence when (i) the subunit bases of a set haveacceptably matched R, Θ, and A values, (ii) subunit backbone structuresare identical or similar in length and (iii) identical or similarsubunit base attachment positions and orientations are used for allsubunits of the set.

Each subunit of such a matched set consists of a subunit base linked ata standard position to a standard-length backbone structure. The subunitbase of each subunit of the set has an R, Θ, and A value closely matchedto the R, Θ, and A values of the subunit bases of the other subunits ofthat set.

According to one feature of the present invention, the polymer subunitsin a set contain at least two different subunit types, each specific fora different oriented base-pair. Specifically, the base of each of atleast two different subunits of the set is effective to form at leasttwo hydrogen bonds with the major-groove sites of its respective targetbase-pair, where one of those hydrogen bonds is to the purine N7nitrogen of the target base-pair, as discussed above.

Other subunit or other subunits in the set may bind, but are notrequired to bind, with high specificity to oriented base-pairs in thetarget sequence. Thus, another subunit of the set may bindsatisfactorily to two different oriented base-pairs, as will be seenbelow. Such low-specificity or non-specific subunits serve to provide(a) required spacing between high-specificity subunits in the polymerand (b) contribute to stacking interactions between the planar bases inthe polymer/duplex complex.

In addition, the subunits in the polymer provide high-specificity basebinding to at least about 70% of the oriented base-pairs in the targetsequence. Thus, where a subunit set includes only two high-specificitybases, the target duplex sequence contain at least 70% orientedbase-pairs which are specifically bound by those two high-specificitybases.

1. Basic Subunit Set for C:G and T:A or U:A Oriented Base-pairs. Themost basic subunit set is suitable for targeting duplex geneticsequences containing only C:G and T:A or U:A oriented base-pairs.

The first member of this basic subunit set is a high-specificity subunitcontaining a guanine, 6-thioguanine, or analog thereof, subunit baseeffective to hydrogen bond specifically to a C:G oriented base-pair. Asillustrated in FIG. 10A, guanine (or 6-thioguanine) forms three hydrogenbonds to the polar major-groove sites of a C:G oriented base-pair,including the guanine N7 of that target base-pair. The subunit may beformed with any of a variety of deoxyribose, ribose or morpholinobackbone structures, with the base attached to the backbone structure inthe β-stereochemical orientation, as illustrated in Example 2.

The second member of the basic set is a high-specificity subunitcontaining a 2,6-diaminopurine, or analog thereof, subunit baseeffective to hydrogen bond specifically to a T:A or U:A orientedbase-pair. As illustrated in FIG. 10B, the 2,6-diaminopurine base formsthree hydrogen bonds to the polar major-groove sites of a T:A or U:Aoriented base-pair, including the adenine N7 of that target base-pair.As with the guanine subunits, a variety of diaminopurine subunits, andanalogs thereof, with deoxyribose, ribose and morpholino backbonestructures, and having the desired β-stereochemical attachment of thebase to the backbone structure, can be prepared by modifications ofavailable nucleosides, also as illustrated in Example 2.

CPK molecular modeling showed that the guanine and diaminopurinemoieties should effectively and specifically bind their targetbase-pairs. Additional support for this major-groove hydrogen-bondingmode was obtained from a best fit analysis carried out for these twotrimolecular complexes, C:G:G and U:A:D. Voet and Rich (1970) tabulatethe lengths and angles of hydrogen-bonds from x-ray diffraction studiesof crystalline complexes of purines and pyrimidines. In thosetabulations NH:N bonds range in length from 2.75 A to 3.15 A and theirangles range from 115° to 145° . NH:O bonds range in length from 2.60 Ato 3.20 A and their angles range from 110° to 145°.

In the best fit calculations, structural parameters used for the purinesand pyrimidines in the Watson-Crick base-pairs are those given by Richand Seeman (1975). Those parameters were obtained from x-ray diffractionof ApU and GpC crystals (right handed anti-parallel Watson-Crick) whichwere solved at atomic resolution. The guanine structural parametersreferenced above were also used for the subunit base in FIG. 10A. The2,6-diaminopurine subunit base of FIG. 10B was assumed to havestructural parameters essentially identical to those of9-ethyl-2,6-diaminopurine obtained from x-ray diffraction studies ofcrystalline trimolecular complexes of 9-ethyl-2,6-diaminopurinehydrogen-bonded to two 1-methylthymines (one thymine bonded in theWatson-Crick mode and the other thymine bonded in the reverse-Hoogsteenmode) as reported by Sakore et al. (1969).

To simplify the analysis, the approximation was made that all atoms arein the same plane. Table 4 gives the results of this analysis. In thistable the standard purine and pyrimidine numbering system is usedthroughout, subunit base-G stands for the subunit base of FIG. 10A(guanine) and subunit base-D for the subunit base of FIG. 10B(2,6-diaminopurine). Angles are measured as in Voet and Rich (1970).

                  TABLE 4                                                         ______________________________________                                                           angle length                                               ______________________________________                                        Guanine subunit base H-bonded to a C:G base-pair                              W/C hydrogen-bonds                                                            O2(C):NH2(G)         125°                                                                           3.17 A                                           N3(C):NH1(G)         119°                                                                           2.95 A                                           NH4(C):O6(G)         129°                                                                           2.63 A                                           Major-Groove hydrogen-bonds                                                   NH2(subunit base-G):N7(G)                                                                          140°                                                                           3.12 A                                           NH1(subunit base-G):O6(G)                                                                          115°                                                                           2.74 A                                           O6(subunit base-G):NH4(C)                                                                          143°                                                                           2.63 A                                           Diaminopurine subunit base H-bonded to a U:A base-pair                        W/C hydrogen-bonds                                                            NH3(U):N1(A)         119°                                                                           2.98 A                                           O4(U):NH6(A)         126°                                                                           2.71 A                                           Major-Groove hydrogen-bonds                                                   NH2(subunit base-D):N7(A)                                                                          137°                                                                           2.85 A                                           N1(subunit base-D):NH6(A)                                                                          139°                                                                           2.95 A                                           NH6(subunit base-D):O4(U)                                                                          132°                                                                           3.00 A                                           ______________________________________                                    

As can be seen from this table, all hydrogen-bond angles and lengths inthe subunit base/base-pair complexes fall within established angle andlength limits for hydrogen-bonds.

A practical difficulty one encounters with polymers consisting of all ornearly all guanine and diaminopurine bases is that in neutral aqueoussolution they undergo self aggregation, which renders them lessavailable for binding their selected duplex target sequences. This selfaggregation probably involves tetramer formation wherein the guaninebases are hydrogen bonded by the mode illustrated in FIG. 10C, and thediaminopurine bases may be hydrogen bonded by the mode illustrated inFIG. 10D.

This self aggregation problem can be alleviated by replacing some of theguanine and/or diaminopurine bases (preferably at least 20%) withguanine and/or diaminopurine analogs which do not have a hydrogen bondacceptor site at the 7 position. This can be achieved in a variety ofways, including the use of a carbon (as in 7-deazaguanine-containingsubunits; Seela 1981; Winkeler 1983) or a methylated nitrogen at the 7position of the guanine and/or diaminopurine base. Several preferredguanine analogs which lack a hydrogen bond acceptor site at the 7position are shown in FIG. 10E. Related diaminopurine analogs also serveto prevent self aggregation.

2. Spacer Subunits for A:T and G:C Oriented Base-pairs. The basic"guanine plus diaminopurine" subunit set can be easily prepared fromguanosine, or deoxyguanosine, and from analogs thereof lacking ahydrogen bond acceptor site at the 7 position, such as 7-deazaguanosine.However, binding polymers assembled from only these two subunits, andtargeted against sequences of at least 16 contiguous base-pairs, areexpected to have targets in only large viruses having genome sizes onthe order of 65,000 base-pairs or greater.

However, it is desirable to have binding polymers which can be targetedagainst a much broader range of viruses, including even small viruses,such as Hepatitis B which has a genome size of only 3,200 base-pairs.One effective approach to extending the targeting range of these bindingpolymers is to target sequences composed predominantly (at least about70%) of target base-pairs for the guanine and diaminopurine (and analogsthereof) high-specificity subunit bases (ie., oriented base-pairs C:Gand T:A or U:A). The remaining base-pairs in the target sequence (i.e.,no more than about 30% G:C and/or A:T or A:U) can then be accommodatedby low-specificity "spacer" bases in the binding polymer, which serveprimarily to provide continuity of stacking interactions between thecontiguous subunit bases of the binding polymer when that polymer is inposition on its target duplex.

Thus, in one embodiment, a polymer assembled from the basic subunit set,described in Section F1, additionally includes one or morelow-specificity spacer subunit bases.

When the binding polymer is in position on its target duplex, with thesubunit bases stacked, the spacer subunit bases (which are notnecessarily hydrogen-bonded to their respective base-pairs) should haveR, Θ, and A values which can closely match the R, Θ, and A values of thehigh-specificity subunit bases. Specifically, for the full subunit set,the R values should all be within about 2 Å, Θ values should all bewithin about 20° , and A values should all be within about 30°.Preferably, the spacer subunit bases should also provide modesthydrogen-bonding to their respective target base-pairs so as to makesome contribution to target binding specificity and affinity.

Where the target sequence contains a G:C oriented base-pair, onepreferred spacer subunit in the subunit set contains a cytosine base,which can hydrogen-bond weakly to G:C and to T:A oriented base-pairs.FIG. 11A shows cytosine hydrogen bonded to the major-groove sites of aG:C base-pair, and FIG. 11B shows cytosine hydrogen bonded to a T:Abase-pair. In neither case does this include a hydrogen bond to the N7of the purine of a target base-pair.

Where the target sequence contains an A:T or A:U oriented base-pair, onepreferred spacer subunit in the subunit set contains a uracil (orthymine) base, which can hydrogen-bond weakly to A:T and to C:G orientedbase-pairs. FIG. 12A shows uracil hydrogen bonded to the major-groovesites of an A:T base-pair, and FIG. 12B shows uracil hydrogen bonded toa C:G base-pair. As with the cytosine spacer, neither of these hydrogenbonding interactions involve the N7 of the purine of a target base-pair.

Although these two subunit spacer bases provide only low-specificity andlow affinity binding to their target base-pairs, nonetheless: i) theyeffectively provide for continuity of subunit base stacking in thetarget-bound binding polymer; ii) they have R, Θ, and A values which areacceptably matched with the R, Θ, and A values of the high-specificityguanine and diaminopurine subunit bases of the subunit set; and iii) thespacer subunits, or close precursors thereto, are commercially availableand relatively inexpensive.

Syntheses of subunit sets containing the four subunit bases guanine,diaminopurine, cytosine, and uracil (or thymine), and having variousdeoxyribose, ribose and morpholino backbone structures, are described inExample 2. The sets described in the example have the following backbonestructures:

A. 2'-deoxyribose, seen in FIG. 5A, Example 2A;

B. 2'-O-methylribose, seen in FIG. 5B (R=methyl), Example 2B;

C. morpholino, seen in FIG. 6A, Example 2C;

D. N-carboxymethylmorpholino-5'-amino, seen in FIG. 6C, Example 2D;

E. N-carboxymethylmorpholino-(alpha)5'-amino, seen generally in FIG. 6F,Example 2E;

F. ribose with 5'carbazate, seen in FIG. 5C, Example 2F;

G. ribose with 5'sulfonylhydrazide, seen in FIG. 5C, but where thecarbonyl group is replaced by a sulfonyl group, Example 2G;

H. ribose with 5'glycinamide, seen in FIG. 5C, but where the OCONHNH₂group is replaced by NHCOCH₂ NH₂, Example 2H; and,

I. ribose with 5'(aminomethyl)(ethyl)phosphate, seen in FIG. 5C, butwhere the OCONHNH₂ group is replaced by OPO₂ EtCH₂ NH₂, Example 2I.

Table 5 shows the base-pair specificities and approximate R, Θ, and Avalues for the subunit bases of this guanine, diaminopurine, cytosine,and uracil (or thymine) subunit set.

                  TABLE 5                                                         ______________________________________                                        Subunit Base                                                                            Base-pair Specificity                                                                        R.sub.a  Θ.sub.a                                                                      A                                      ______________________________________                                        G         C:G            5.8 A    33°                                                                         60°                             D         T:A            5.6 A    32°                                                                         60°                             C         G:C & T:A      4.8 A    38°                                                                         50°                             U         A:T & C:G      4.8 A    38°                                                                         50°                             ______________________________________                                    

Binding polymers prepared with the above G, D, C and U or T subunit setalso have the potential to bind to single-stranded genetic sequences.Specifically, the polymer will be able to bind in a Watson/Crick pairingmode to a single-stranded polynucleotide of the appropriate basesequence.

Since the spacer subunits, C and U or T, in the polymer are degeneratein binding specificity, at least two of these low-specificity spacersubunits are required to provide a level of target specificityequivalent to that provided by one high-specificity subunit. Thus, abinding polymer containing 16 high-specificity subunit bases providesabout the same level of target specificity as a binding polymercontaining 12 high-specificity subunit bases and 8 low-specificityspacer subunit bases.

3. Subunit Set with a Tautomeric Subunit Specific for A:T and G:COriented Base-pairs. In another embodiment, the "guanine plusdiaminopurine" subunit set described in Section F1 includes anadditional subunit having a tautomeric subunit base capable of hydrogenbonding to either G:C or A:T oriented base-pairs. A generalized skeletalring structure and hydrogen bonding array of one preferred base type isshown in FIG. 13A, where X₁ is H or NH₂ ; X₂ is H, F, or C; and Bindicates the polymer backbone. FIGS. 13B-13D show three preferredembodiments of this tautomeric base, as discussed further below.

The hydrogen bonding to target base-pairs by different tautomeric formsof the base from FIG. 13D is shown in FIGS. 14A and 14B for G:C and A:Toriented base-pairs, respectively. As seen from FIG. 14, X₂ can behydrogen-bond acceptor when the tautomer is hydrogen bonded to a G:Cbase-pair, to provide three hydrogen bonds to the base-pair. Similarly,X₁ can be a hydrogen-bond donor when the tautomer is hydrogen bonded toan A:T base-pair, to provide three hydrogen bonds to the base-pair.

Table 6 shows the base-pair specificities and approximate R, Θ, and Avalues for the subunit bases of the guanine, diaminopurine, and thesubunit base of FIG. 14:

                  TABLE 6                                                         ______________________________________                                        Subunit Base                                                                              Base-pair Specificity                                                                       R.sub.a  Θ.sub.a                                                                      A                                     ______________________________________                                        G           C:G           5.8 A    33°                                                                         60°                            D           T:A           5.6 A    32°                                                                         60°                            Tautomeric Base                                                                           G:C & A:T     6.3 A    36°                                                                         55°                            of FIG. 13B                                                                   ______________________________________                                    

The syntheses of a number of specific embodiments of a tautomericsubunit are described in Example 3. The synthesis of the structures seenin FIG. 13B and 13C are described in Example 3A for the 2'doexyribosebackbone structure; in Example 3B for the 2'O-methylribose backbone; andin Example 3C for the morpholino backbone.

4. Subunit Set with High-Specificity Subunits for A:T and G:C OrientedBase-pairs. In another embodiment, the "guanine plus diaminopurine"subunit set described in Section F-1 includes an additional subunitwhose base is specific for hydrogen bonding to a G:C oriented base-pair,or an additional subunit whose base is specific for hydrogen bonding toan A:T (or A:U) oriented base-pair, or the set includes two additionalsubunits whose bases are specific for hydrogen bonding to a G:C orientedbase-pair and to an A:T or A:U oriented base-pair, respectively.

FIG. 15A shows the skeletal ring structure and hydrogen bonding array ofa general type of base effective to bind a G:C oriented base-pair. Threepreferred embodiments of this structure type are shown FIGS. 15B-15D.FIG. 16 shows the structure in FIG. 15D hydrogen-bonded to its G:Ctarget base-pair. As seen from FIG. 15A and FIG. 16, the X₂ position inthe FIG. 15A structure may be a hydrogen bond acceptor, e.g., O, forforming three hydrogen bonds between the base and its target G:Cbase-pair.

Syntheses for subunits having a morpholino backbone structure and theG:C-specific bases of FIGS. 15B and 15C are described in Example 4D.

FIG. 17A shows the skeletal ring structure and hydrogen bonding array ofanother general type of base effective to bind a G:C oriented base-pair.A preferred embodiment of this structure type hydrogen-bonded to its G:Ctarget base-pair is shown in FIG. 17C.

Synthesis of a subunit having a morpholino backbone structure and theG:C-specific base of FIG. 17B is described in Example 4D.

FIG. 18A shows the skeletal ring structure, hydrogen bonding array, andbackbone attachment position of still another general type of baseeffective to bind a G:C oriented base-pair. Several preferredembodiments of this structure are shown in FIGS. 18B-18D. FIG. 19 showsone embodiment of the 18A structure (18B) hydrogen bonded to its G:Ctarget base-pair.

Synthesis of a subunit having a morpholino backbone structure and theG:C-specific base of FIG. 18B is described in Example 4D.

FIG. 20A shows the skeletal ring structure, hydrogen bonding array, andbackbone attachment position of a general type of base effective to bindan A:T or A:U oriented base-pair. Three preferred embodiments of thisstructure type are shown in FIGS. 20B-20D. FIG. 21 shows the structurein FIG. 20D hydrogen-bonded to its A:T target base-pair.

Syntheses for subunits having a morpholino backbone structure and theA:T or A:U-specific bases of FIGS. 20B and 20C are described in Example4C.

FIG. 22A shows the skeletal ring structure, hydrogen bonding array, andbackbone attachment position of still another general type of baseeffective to bind an A:T or A:U oriented base-pair. Several preferredembodiments of this structure are shown in FIGS. 22B-22D. FIG. 23 showsone embodiment of the 22A structure (22B) hydrogen bonded to its A:Ttarget base-pair.

Synthesis of a subunit having a morpholino backbone structure and theA:T-specific base of FIG. 22B is described in Example 4C.

The subunits described in this section whose bases are specific for G:C,A:T and A:U oriented base-pairs, with the guanine and diaminopurinesubunits described in Section F1, provide a complete set of subunitsproviding high-specificity hydrogen bonding for each of the fourpossible oriented base-pairs in duplex nucleic acids. A subunit setformed in accordance with one aspect of the invention may include anythree of these high-specificity subunits effective to bind to threedifferent oriented base-pairs in a duplex target sequence. For example,in a target sequence containing T:A, C:G, and G:C base-pairs, theselected subunit set would include three different subunits containing acommon or similar backbone structure and diaminopurine, guanine (orthioguanine), and one of the above G:C-specific bases. A subunit setsuitable for a target sequence containing all four oriented base-pairswould additionally include a subunit whose base is one of the abovehigh-specificity bases for an A:T or A:U oriented base-pair.

Table 7 shows the base-pair specificities and approximate R_(b), Θ_(b),and A values for the subunit bases comprising guanine, diaminopurine,and the high-specificity bases of FIGS. 15, 17A, 17B, 18, 20, and 22.

                  TABLE 7                                                         ______________________________________                                        Subunit Base                                                                              Base-pair Specificity                                                                       R.sub.b   Θ.sub.b                                                                     A                                     ______________________________________                                        G           C:G           10.0 A    19°                                                                        60°                            D           T:A           9.9 A     19°                                                                        60°                            Base of FIG. 15                                                                           G:C           10.2 A    22°                                                                        55°                            Base of FIG. 17A                                                                          G:C           8.8 A     19°                                                                        50°                            Base of FIG. 17B                                                                          A:T           8.8 A     19°                                                                        50°                            Base of FIG. 18                                                                           G:C           9.3 A     21°                                                                        40°                            Base of FIG. 20                                                                           A:T           10.3 A    20°                                                                        55°                            Base of FIG. 22                                                                           A:T           9.3 A     21°                                                                        40°                            ______________________________________                                    

This table illustrates the general suitability of this set of bases inregard to R, Θ, and A values.

5. High-Specificity Subunit Set with Replacements for Guanine andDiaminopurine. When guanine and diaminopurine bases are hydrogen bondedto the polar major-groove sites of their respective target base-pairs ina duplex DNA sequence existing in a B conformation, those two basesafford less than optimal stacking interactions between contiguous basesof the binding polymer. Since stacking interactions generally contributesignificantly to the binding affinity of these polymers for their duplexgenetic targets, use of bases affording improved stacking interactionscan yield polymers with greater target binding affinities.

Accordingly, in another embodiment, the binding polymer includes one ormore subunits having bases which are specific for C:G and/or T:A or U:Awhich provide better stacking interactions than afforded by guanineand/or diaminopurine, particularly when the polymer is bound to itstarget genetic duplex in a B or B-like conformation.

One preferred subunit set which can be used to prepare polymers withimproved stacking interactions when targeted against B-form geneticduplexes includes two or more subunits selected from the following: aG:C-specific base illustrated in FIG. 18; an A:T or A:U-specific baseillustrated in FIG. 22; plus an additional acceptably matched (withrespect to R, Θ, and A values) subunit whose base is specific forhydrogen bonding to a C:G oriented base-pair and which affords betterstacking interactions than guanine; and, an additional acceptablymatched subunit whose base is specific for hydrogen bonding to an A:T orA:U oriented base-pair and which affords better stacking interactionsthan diaminopurine.

FIG. 24A illustrates the general skeletal ring structure, hydrogenbonding array, and backbone attachment position of such an acceptablymatched base designed for binding to a C:G oriented base-pair, and FIG.24C illustrates a specific embodiment of the 24A structure. FIG. 24Billustrates the general skeletal ring structure, hydrogen bonding array,and backbone attachment position of a related base also designed forbinding to a C:G oriented base-pair, and FIG. 24D illustrates a specificembodiment of the 24B structure.

Synthesis of subunits having a morpholino backbone structure and theC:G-specific bases of FIG. 24C and 24D are described in Example 4A.

FIG. 25 shows the hydrogen bonding of the 24C structure to a C:Goriented base-pair.

FIG. 26A illustrates the general skeletal ring structure, hydrogenbonding array, and backbone attachment position of such an acceptablymatched base designed for binding to a T:A or U:A oriented base-pair,and FIG. 26C and 26D illustrate two specific embodiments of the 26Astructure. FIG. 26B illustrates the general skeletal ring structure,hydrogen bonding array, and backbone attachment position of a relatedbase also designed for binding to a T:A or U:A oriented base-pair, andFIG. 26E illustrates a specific embodiment of the 26B structure.

Synthesis of subunits having a morpholino backbone structure and the T:Aor U:A-specific bases of FIG. 26D and 26E are described in Example 4B.

FIG. 27 shows the hydrogen bonding of the 26C structure to a T:Aoriented base-pair.

The subunits described in this section provide a complete set ofsubunits affording high-specificity hydrogen bonding for each of thefour possible oriented base-pairs in duplex nucleic acids, as well asimproved stacking interactions between contiguous bases when the bindingpolymer is in position on its target genetic duplex, especially whenthat target duplex is in a B or B-like conformation.

Binding polymers containing subunits of this set may also include one ormore guanine and/or diaminopurine bases, or analogs thereof.

Table 8 shows the base-pair specificities and approximate R_(b), Θ_(b),and A values for the subunit bases comprising guanine, diaminopurine,and the bases of FIGS. 18, 22, 24 and 26.

                  TABLE 8                                                         ______________________________________                                        Subunit Base                                                                             Base-pair Specificity                                                                       R.sub.b   Θ.sub.b                                                                      A                                     ______________________________________                                        G          C:G           10.0 A    19°                                                                         60°                            Base of FIG. 24                                                                          C:G           9.6 A     16°                                                                         50°                            D          T:A           9.9 A     19°                                                                         60°                            Base of FIG. 26                                                                          T:A           9.6 A     15°                                                                         50°                            Base of FIG. 18                                                                          G:C           9.3 A     21°                                                                         40°                            Base of FIG. 22                                                                          A:T           9.3 A     21°                                                                         40°                            ______________________________________                                    

6. Additional High-Specificity Subunit Set with Replacements for Guanineand Diaminopurine. Another preferred subunit set which can be used toprepare polymers with improved stacking interactions when targetedagainst B-form genetic duplexes includes the following.

FIG. 28A illustrates the general skeletal ring structure, hydrogenbonding array, and backbone attachment position of a base designed forbinding to a T:A or U:A oriented base-pair, and FIG. 28B illustrates aspecific embodiment of the 28A structure.

Synthesis of a subunit having a morpholino backbone structure and theT:A or U:A-specific base of FIG. 28B is described in Example 4B.

FIG. 29A illustrates the general skeletal ring structure, hydrogenbonding array, and backbone attachment position of a base designed forbinding to a C:G oriented base-pair, and FIG. 29B illustrates a specificembodiment of the 29A structure.

Synthesis of a subunit having a morpholino backbone structure and theC:G-specific base of FIG. 29B is described in Example 4A.

FIG. 30A illustrates the general skeletal ring structure, hydrogenbonding array, and backbone attachment position of such an acceptablymatched base designed for binding to a A:T or A:U oriented base-pair,and FIGS. 30C-30E illustrate three specific embodiments of the 30Astructure. FIG. 30B illustrates the general skeletal ring structure,hydrogen bonding array, and backbone attachment position of a relatedbase also designed for binding to a A:T or A:U oriented base-pair, andFIG. 30F illustrates a specific embodiment of the 30B structure.

Synthesis of subunits having a morpholino backbone structure and the A:Tor A:U-specific bases of FIGS. 30C and 30F are described in Example 4C.

FIG. 31A illustrates the general skeletal ring structure, hydrogenbonding array, and backbone attachment position of such an acceptablymatched base designed for binding to a G:C oriented base-pair, and FIGS.31C illustrates a specific embodiment of the 31A structure. FIG. 31Billustrates the general skeletal ring structure, hydrogen bonding array,and backbone attachment position of a related base also designed forbinding to a G:C oriented base-pair, and FIG. 31D illustrates a specificembodiment of the 31B structure.

Synthesis of subunits having a morpholino backbone structure and theG:C-specific bases of FIGS. 31C and 31D are described in Example 4D.

FIG. 32 illustrates binding of representative bases from this subunitset to their respective target base-pairs, including: the FIG. 28Bstructure hydrogen bonded to a U:A oriented base-pair; the FIG. 29Bstructure hydrogen bonded to a C:G oriented base-pair; the FIG. 30Dstructure hydrogen bonded to an A:T oriented base-pair; and, the FIG.31C structure hydrogen bonded to a G:C oriented base-pair.

The subunits described in this section provide a complete set ofsubunits affording high-specificity hydrogen bonding for each of thefour possible oriented base-pairs in duplex nucleic acids, as well asimproved stacking interactions between contiguous bases when the bindingpolymer is in position on its target genetic duplex, especially whenthat target duplex is in a B or B-like conformation.

Table 9 shows the base-pair specificities and approximate R_(b), Θ_(b),and A values for the subunit bases comprising the bases of FIGS. 28, 29,30, and 31.

                  TABLE 9                                                         ______________________________________                                        Subunit Base                                                                             Base-pair Specificity                                                                        R.sub.b  Θ.sub.b                                                                      A                                     ______________________________________                                        Base of FIG. 28                                                                          U:A            9.0 A    6°                                                                          20°                            Base of FIG. 29                                                                          C:G            9.0 A    7°                                                                          20°                            Base of FIG. 30                                                                          A:T            9.8 A    4°                                                                          20°                            Base of FIG. 31                                                                          G:C            9.8 A    5°                                                                          20°                            ______________________________________                                    

II. Polymer Preparation

This section describes assembly of the subunits, described above, into asequence-specific duplex-binding polymer.

A. Polymer Sequence and Length

One use of the polymers of the present invention is to bind to andinactivate a target duplex sequence, such as a sequence essential forpathogenicity of a disease-causing agent, without inactivating normalhost genetic sequences. Thus, the sequence information recognized by thepolymer should distinguish the pathogen sequence from normal hostsequences.

An estimate of the amount of sequence information which a duplex nucleicacid-binding polymer should recognize in a disease-specific sequence inorder to avoid concomitant attack on normal cellular sequences can becalculated as follows. The human genome contains roughly 3 billionbase-pairs of unique-sequence DNA. For a gene-inactivating agent to havean expectation of having no fortuitous target sequences in a cellularpool of 3 billion base-pairs of unique sequence genetic material, itshould recognize at least n base-pairs in its target, where n iscalculated as 4^(n) =3×10⁹. This calculation gives an expected minimaltarget recognition sequence of approximately 16 base-pairs. Accordingly,a gene-inactivating polymer recognizing in excess of 16 base-pairs inits target sequence has a low likelyhood of having no fortuitous targetsin the cellular pool of inherent DNA. Obviously as the number ofbase-pairs recognized in the target sequence increases over this valuethe probability that the polymer will attack inherent cellular sequencescontinues to decrease: as the number of base-pairs recognized by thegene-inactivating polymer increases linearly, this "safety factor"increases exponentially.

To illustrate, Table 8 tabulates the number of base-pairs recognized ina target sequence and the corresponding expected number of fortuitoustargets in a pool of 3 billion base-pairs of unique-sequence geneticmaterial.

                  TABLE 10                                                        ______________________________________                                        Number of base-pairs                                                                           Expected number of fortuitous                                recognized in target duplex                                                                    targets in human genome                                      ______________________________________                                         8               45,776                                                       10               2,861                                                        12               179                                                          14               11.2                                                         16               0.7                                                          18               0.044                                                        20               0.0027                                                       ______________________________________                                    

The numbers in Table 10 indicate that in order to achieve specificityfor a pathogen or pathogenic state, a binding agent targeted to duplexnucleic acids should recognize at least 16, and preferably 18 or morebase-pairs of the target sequence, to avoid fortuitous sequence binding.

In addition to target sequence length, it is important to consider howmany of the four possible oriented base-pairs in duplex nucleic acids(ie., A:T, C:G, G:C, and T:A) must be specifically recognized by thepolymer bases in order to allow practical targeting of various viralpathogens. Table 9 shows the approximate number of targets expected in arelatively small viral genome (about the size of the HIV provirus) as afunction of the number of different base-pair-binding specificities in a16-subunit polymer. The values in the table were calculated on theassumption that the purine to pyrimidine ratio in a given strand of thepathogen's genome is approximately 1.0 and that the bases areeffectively in a random order.

                  TABLE 11                                                        ______________________________________                                                         Expected number of contiguous                                Number of base-pair-binding                                                                    16-base-pair targets in a                                    specificities in subunit set                                                                   10,000 base-pair viral genome                                ______________________________________                                        1                0.000002                                                     2                0.15                                                         3                100                                                          4                10,000                                                       ______________________________________                                    

The tabulated values demonstrate that, typically, homopolymers (i.e.,polymers assembled from subunits having specificity for just oneoriented base-pair) are unlikely to have targets in natural duplexgenetic sequences. Copolymers of two subunit types, with specificitiesfor only two of the four oriented base-pairs, are expected to havecontiguous 16-base-pair targets in large viruses, such as Herpes SimplexVirus I. In contrast, binding polymers assembled from subunit setshaving specificities for three or four of the oriented base-pairs havean adequate number of targets in even the smallest DNA viruses (e.g.,Hepatitis B with a genome size of only 3200 base-pairs).

As described in Section I, the basic two-subunit set formed inaccordance with the present invention includes two subunits which arespecific for two different oriented base-pairs, C:G and T:A or U:A. Toincrease targeting versatility, polymers can be assembled from anexpanded subunit set which includes one or two spacer subunits inaddition to the specific subunits.

Polymers can be assembled from the basic two-subunit set plus anadditional semi-specific subunit whose base is capable of hydrogenbonding to either of two different oriented base-pairs (for example, thebase of FIG. 13 with A:T or A:U and G:C). As noted above, thissemi-specific subunit base recognizes only half the sequence informationrecognized by a high-specificity subunit base, and thus its use requiresa correspondingly longer polymer in order to achieve adequatespecificity for its target.

Further, polymers can be assembled from three or four different subunitswhose high-specificity bases are each capable of hydrogen bonding tojust one of the four oriented base-pairs. Such a subunit set containingsubunit bases specific for each of the four possible oriented base-pairsallows construction of a polymer targetable against essentially anydesired duplex genetic sequence.

Still another embodiment comprises polymers containing specialmethyl-discriminating subunits. The moiety at the 5 position of thepyrimidine of base-pairs in duplex nucleic acids provides an exploitablestructural difference that yeilds an additional level of targetingspecificity. For example, (i) duplex DNA contains a methyl at the 5position of thymine in A:T and T:A base-pairs, while duplex RNA lacks amethyl at the corresponding position of uracil in A:U and U:Abase-pairs, and (ii) clusters of 5'-CpG sequences in the control regionsof transcriptionally-inactive genes contain methyls at the 5 position ofthe cytosines, whereas corresponding transcriptionally-active genesgenerally lack methyls at the 5 position of these cytosines. The moietyat the 5 position of pyrimidines in duplex nucleic acids has beenexploited for target discrimination in nature, for example, the controlof cleavage by restriction nucleases in bacteria. The polymers of thepresent invention (see Section I-C) provides a means to exploit thesestructural differences to provide a new level of duplex targetdiscrimination. Such polymers provide an additional level of targetdiscrimination by affording effective binding only to duplex targetsequences containing a hydrogen at the 5 position of the pyrimidine ofspecified base-pairs in the target sequence.

B. Subunit Activation and Polymer Assembly

The subunits, prepared as in Examples 1-5, can be activated and thencoupled in a controlled sequential manner to give the desired bindingpolymer. Representative polymer assembly procedures fordeoxyribose-containing and 2'-0-methylribose-containing subunits aredescribed in Example 6. Representative activation procedures formorpholino-containing subunits are described in Example 7; Example 8describes an exemplary procedure for assembling these activated subunitsvia solid-phase stepwise addition to give the desired binding polymers;and, Example 9 describes their purification. FIG. 33 illustrates onesubunit addition cycle of this stepwise assembly procedure using arepresentative morpholino subunit prepared as in Example 2C andactivated as in Example 7A.

FIGS. 34 and 35 illustrate four-subunit-long segments of representativepolymers assembled from the subunit set described in Section 5. I. F4,prepared as in Example 4, and activated as in Example 7A.

FIG. 36 illustrates a 4-subunit-long segment of a representative polymerassembled from the subunit set described in Section F-5, prepared as inExample 4, and activated as in Example 7A.

FIG. 37 illustrates a 4-subunit-long segment of a corresponding polymercontaining two methyl-discriminating subunits which afford binding to anRNA/RNA target sequence, but preclude binding to a corresponding DNA/DNAtarget sequence.

FIG. 38 illustrates a 4-subunit-long segment of a representative polymerassembled from the subunit set described in Section F-5, containing a7-atom unit-length backbone suitable for binding a target duplex in theB conformation, and further containing two methyl-discriminatingsubunits which afford binding to a target sequence containing a CpG/GpCsubsequence, but which preclude binding to a corresponding(5mC)pG/Gp(5mC) subsequence.

C. Novel Polymer Assembly Comprising: Oxidation/Ring Closure/Reduction

In addition to the above, the following exemplary coupling procedure canalso be used for assembling the desired nucleic acids binding polymers(FIG. 39). This assembly procedure involves: (i) providing a subunit, orblock of linked subunits, which contains vicinyl aliphatic hydroxyls,but no free primary amine (e.g., structure 1 of FIG. 39), (ii) oxidizingthose vicinyl hydroxyls to give a dialdehyde component (e.g., structure2 of FIG. 39), (iii) providing a subunit, or block of subunits, whichcontains a free primary aliphatic amine (e.g., structure 3 of FIG. 39,and subunits prepared as in Examples 2F-2I), (iv) contacting thedialdehyde component with the primary amine component to effect couplingof the two components via formation of a cyclic morpholino structurehaving hydroxyls on the carbons adjacent to the morpholino nitrogen(e.g., structure 4 of FIG. 39), and, (v) during or after the couplingreaction, or after completion of polymer assembly, adding a reducingagent to remove the hydroxyls on the carbons adjacent to the morpholinonitrogen, to give the desired morpholino ring structure (e.g., structure5 of FIG. 39).

The vicinyl-hydroxyl-containing moiety can be other than ribose, such asgalactose or glucose. Further, this coupling method can be used ineither a solution-phase or a solid-phase mode for polymer assembly.Also, the oxidation step and the subsequent coupling step are preferablycarried out in alcohol or water or a mixture thereof, and at a pH nearneutrality. Although the reduction can be carried out during or afterthe coupling, best results are obtained when reducing agent, e.g.,NaCNBH₄, is present during the coupling step. Complete reduction anddisruption of borate complexes (generated when NaCNBH₄ is used for thereduction) is best achieved by a final acidic wash having a pH in therange of 3 to 5--which can be carried out after each coupling, or afterall couplings are completed.

Example 10 describes a representative application of this"oxidation/ring closure/reduction" coupling method for stepwisesolid-phase assembly of a binding polymer.

D. Polymer Modifications

If it is necessary or desireable, the solubility of the polymer can beenhanced by addition of a hydrophilic moiety, such as a polyethyleneglycol (PEG) chain. This can be accomplished, according to one approach,by deprotecting the polymer terminus, and reacting the polymer withexcess of activated hydrophilic compound, e.g., PEG activated bybis(p-nitrophenyl)carbonate. Thereafter the binding polymer is cleavedfrom the synthesis support and treated with ammonium hydroxide to removethe base-protecting groups, and then purified, preferably by ionexchange chromatography at pH 10.5. One preferred hydrophilic moleculeis PEG having an average molecular weight of about 1000 daltons(commercially available from Polysciences, Inc. and Aldrich Chem. Co.).

For some applications it may be desirable to modify the polymer to favorits cellular uptake via endocytosis. This may be done, for example, byderivatizing the polymer with a polycationic molecule, such aspolylysine. Coupling of such a polycationic molecule containing one ormore primary amine moieties, may be accomplished by reacting thebase-protected polymer with a bifunctional coupling agent, such asdisuccinimidyl suberate, or other commercially available agent (e.g.,Pierce Chemical Company) and then adding the amine-containingpolycationic molecule.

Where the polymer molecules are to be attached to a solid support, e.g.,in a diagnostic system, the terminal N-protective group can be cleaved(leaving the bases still in the protected state) and reacted with asuitable crosslinking agent, such as disuccinimidyl suberate. Thispreparation is then added to the support material, such as latexmicroparticles containing suitable linker arms terminating in primaryamine moieties.

Alternatively, if it is desired to purify the binding polymer prior toattachment to a support, a methoxytritryl- protected 6-aminocaproic acidcan be linked to the unprotected N-terminus of the binding polymer usingdicyclohexylcarbodiimide (DCC). The binding polymer is then treated withammonium hydroxide to deprotect the bases, purified by standard methods,and the terminal methoxytrityl is cleaved from the aminocaproic acidmoiety. Finally, the purified polymer is mixed with support materialhaving suitable linker arms terminating in p-nitrophenylester moieties,to give covalent coupling of the polymer molecules to the support.

Binding polymers constructed from subunits having cyclic backbonemoieties have a strand polarity analogous to the 5' to 3' strandpolarity exhibited by standard phosphodiester-linked polynucleotides.The binding polymers of the present invention have the potential to bindtheir target duplex in either or both of the two oridentations.Accordingly, for a given heteromeric target sequence of base pairs, twobinding polymers can be constructed, one having the proper sequence ofbases ordered from 5' to 3', and the other having the same sequence ofbases, but ordered 3' to 5'. These two polymers can then be tested fortheir respective binding affinities for the selected duplex targetsequence. Similar approaches for determining proper binding orientationsfor standard polynucleotides are well-known in the art.

E. Polymer Structural Characterization

NMR, two-dimensional NMR, and elemental analysis appears to providelittle useful structural information for heteropolymers of the presentinvention when the polymers are of any significant length (for example,polymers greater than 12 subunits in length).

Polymers prepared as in Example 8 and cleaved from the solid support,but not yet treated with ammonium hydroxide, typically show relativelyclean parent ions for polymers up to about 16 to 18 subunits in length,when assessed by positive fast atom bombardment mass spectrometry. Forlonger polymers, and for polymers lacking protective groups on the bases(such as prepared in Example 10), effective mass analysis requiresprocedures such as laser desorption or electro-spray.

III. Utility

A. Diagnostics: Detection of Sequences in Duplex Form

In one application, the polymer of the invention is used in a diagnosticmethod for detecting a duplex target nucleic acid sequence in ananalyte. The target sequence is typically a pathogen-specific sequence,such as a virus or bacterial genome sequence, which is to be detected ina biological sample, such as a blood sample.

The target sequence is preferably 15 to 25 subunits in length, toprovide the requisite sequence specificity, as discussed above. In oneassay format, the diagnostic reagent is a solid support, such as amicro-bead, coated by covalently-bound polymers effective tospecifically bind to the duplex target sequence. After sample treatmentto release the analyte duplex from bacterium or virus in free form, ifnecessary, the sample is contacted with the solid support underconditions sufficient to effect base-pair-specific binding of theanalyte duplex to the support-bound polymer. Typically, the bindingreaction is performed at 20°-37° C. for 10 minutes to 2 hours. Afterwashing the solid support to remove unbound material, the support iscontacted with a reporter reagent effective to bind to the capturedtarget duplex, to allow detection of said duplex. The reporter may be asoluble duplex-binding polymer, formed in accordance with the presentinvention, which is base-pair-specific for a second analyte-specifictarget sequence in the analyte duplex, and which is labeled with asuitable signal group, such as a fluorescent moiety, for signaldetection. The signal group is coupled to the polymer by standardcoupling methods, such as described in Section II.

After washing the support, it is examined for bound reporter, which willbe proportional to the amount of analyte bound to the support via thesequence-specific binding polymer.

Alternatively, the washed support containing bound analyte duplex may bereacted with a fluorescent intercalating agent specific for nucleicacids, such as ethidium bromide, and then the polymer-bound analyte isassessed by its fluorescence. Another alternative is to react the washedsupport containing bound analyte duplex with a reporter-labeledpolycationic molecule, such as a fluorescent-labeled oligo-cation, asdescribed in co-owned published PCT Application No. PCT/US86/00545 (WO86/05519). The reporter molecule binds by electrostatic interactionswith the negatively charged analyte duplex backbone, but does not bindthe substantially uncharged polymer molecules on the solid support.After washing the support to remove unbound material, the reporter boundto the solid support, via the sequence-specific analyte/polymer complex,is measured.

B. In situ Hybridization

In many applications, the in situ hybridization is directed toward atarget sequence in a double-stranded duplex nucleic acid, typically aDNA duplex associated with a pathogen or with a selected sequence inchromosomal DNA. To date, in situ hybridization typically includesaddition of a labeled nucleic acid probe to a permeabilized cellstructure, the structure is heated to a temperature sufficient todenature the target duplex nucleic acid, and the probe and denaturednucleic acid are allowed to react under suitable hybridizationconditions. After removing unbound (non-hybridized) probe, the structureis examined for the presence of reporter label, allowing the site(s) ofprobe binding to target nucleic acid to be localized in the biologicalstructure.

This method has been widely applied to chromosomal DNA, for mapping thelocation of specific gene sequences and determining distances betweenknown gene sequences, for studying chromosomal distribution of satelliteor repeated DNA, for examining nuclear organization, for analyzingchromosomal aberrations, and for localizing DNA damage in single cellsor tissue. Several studies have reported on the localization of viralsequences integrated into host-cell chromosomes. The method has alsobeen used to study the position of chromosomes, by three-dimensionalreconstruction of sectioned nuclei, and by double in situ hybridizationwith mercurated and biotinylated probes, using digital image analysis tostudy interphase chromosome topography (Emmerich). Another generalapplication of the in situ hybridization method is for detecting thepresence of virus in host cells, as a diagnostic tool.

In the present application, the polymer of the invention is designed fortargeting a specific duplex genetic sequence associated with a cellularor subcellular structure of interest, such as a chromosomal preparation.The polymer is derivatized with a suitable label such as a fluorescenttag. The polymer is preferably added directly to cells or tissuecontaining the structure being studied, without first permeabilizing thematerial. Because the polymer is uncharged it can more readily penetrateinto living cells without the need for a permeabilization treatment. Itfurther offers the advantage of being resistant to nuclease degradation.

Once in contact with the duplex target material of interest,base-pair-specific binding can occur at normal physiologicaltemperatures, again allowing detection of duplex targets underconditions of normal cell activity, and without heat disruption of thematerial being studied. After a time sufficient for binding to thetarget duplex, and washout of unbound polymer, the structure beingstudied may be examined directly, e.g., by fluorescence microscopy, toobserve site-specific localization of the duplex target sequence andpossible movement thereof. Alternatively, to reduce fluorescencebackground, the material may be fixed, e.g., by ethanol treatment,washed to remove unbound reporter, and viewed in fixed form bymicroscopy.

C. Isolation of Duplexes Containing Target Sequence

Another general application of the polymer invention is for isolatingduplex nucleic acid structures from a nucleic acid mixture, such as amixture of genomic fragments, a blood sample containing a selected viralduplex, or a mixture of plasmids with different duplex inserts indifferent orientations.

The binding polymer used in the method is (a) designed forbase-pair-specific binding to a selected target duplex sequence and (b)capable of being isolated from a liquid sample after capture of thetarget duplex. To this end, the polymer may be bound to a solid support,as described above, or may be derivatized with a ligand moiety, such asbiotin, which permits capture on a solid support, orimmunoprecipitation, after binding to the target duplex.

The polymer is added to the sample material and incubated underconditions which allow binding of the polymer to its target sequence,typically for 10 minutes to 2 hours at 20°-37° C. After binding hasoccurred, the polymer and bound material is isolated from the sample.The isolated material may be released from the polymer by heating, or bychaotropic agents, and further amplified, if necessary by polymerasechain reaction methods, and/or clonal propagation in a suitable cloningvector.

D. Site-Specific DNA Modification

The polymer of the invention is also useful for producing selectedsite-specific modifications of duplex DNA in vitro. These may includecutting a duplex species at a selected site, or protecting a selectedregion against restriction or methylating enzymes. The latterapplication is useful particularly in recombinant DNA technology, whereit is often advantageous to be able to protect a vector or heterologousDNA sequence against cutting by a selected restriction endonuclease, orwhere it is desired to selectively prevent methylation at a givenrestriction site.

To produce site-specific cleavage in a selected base sequence, thepolymer is derivatized with a cleaving moiety, such as a chelated irongroup (Dreyer et al., 1985; Dervan, 1986; Moser et al., 1987; Maher etal., 1989) capable of cleaving duplex DNA in a polymer bound state. Thepolymer sequence is selected to place the cleaving group, which istypically coupled at one polymer end, adjacent to the site to becleaved. To protect a selected region of duplex target sequence againstrestriction or methylase enzymes, the polymer includes a sequence forbinding to the 4-8 base-pair sequence which specifies a selectedrestriction enzyme sequence--plus any additional proximal baseseffective to give increased specificity for a unique target sequence.After addition of the polymer to the duplex material, the material istreated with the selected restriction or methylating enzyme. Afterenzyme treatment, the treated duplex is "deprotected" by heating.

E. Therapeutic Application

The polymers of the invention, by their ability to bind to duplex targetsequences, have the potential to inactivate or inhibit pathogens orselected genomic sequences, such as oncogenes, associated with disease.Origins of replication and enhancer and promoter sequences areparticularly sensitive to inactivation by duplex-directed bindingagents, because the agent can occupy a target site required forinitiation of replication or transcription of the targeted gene. Suchgene-control sequences are known for many pathogenic genes, and also fora variety of oncogenes which have been characterized in humans.

For some therapeutic applications, it may be desirable to modify thebinding polymer to favor its delivery to certain cells or tissues, or tofavor its delivery to certain subcellular organelles, such as thenucleus (Chelsky). This can be accomplished, for example, by linking thebinding polymer to a suitable signal structure, such as desialylatedgalactosyl-containing proteins (Gregoriadis, 1975) or a cluster ofgalactose moieties, which favors uptake by liver cells; or such asD-mannose or L-fucose, which favor uptake by Kupffer cells andmacrophages; or such as insulin or related peptides, which may then beactively transported across the blood/brain barrier. Additionally, thebinding polymers can be incorporated into surfactant micelles, with orwithout brain-specific antibodies, to enhance delivery across theblood/brain barrier (Kabanov).

For the reasons discussed above, the polymer should generally contain atleast 16 base-pair-specific subunits, to minimize the possibility ofundesired binding to sequences other than the intended target sequence.Candidate target structures can be determined from analysis of genomicsequences, such as are available in a variety of sequence databases.Preferred target structures are those which are (a) well conservedacross strains, and (b) have a base-pair sequence which is compatiblewith the set of subunits available for forming the polymer. For example,if the subunit set includes a guanine, diaminopurine, and one or twospacer subunits, as detailed in Section I, the target sequencepreferably contains at least about 70% C:G and T:A oriented base-pairs,and the remainder G:C and/or T:A.

One therapeutic target for the polymers of the present invention is theHIV-I genome. A sequence search of the HIV-I genome in the duplexproviral stage was performed for sequences (i) well conserved acrossstrains and (ii) suitable targets for binding polymers assembledpredominantly from guanine and 2,6-diaminopurine-containing subunits.Table 12 shows several such selected target sequences, and positionedthereon, binding polymers assembled from the "two subunits plus spacers"set of the type described in Section I, E-2.

                                      TABLE 12                                    __________________________________________________________________________    Position                                                                      in Genome                                                                           Gene                                                                              Polymer/Target Complex                                              __________________________________________________________________________              DDDDDUGDUDGGGGGDD        Polymer                                    *-----*---------------------                                                  2431  Pol 5'-AAAAATGATAGGGGGAA     Target                                               TTTTTACTATCCCCCTT-5'     Duplex                                               DUDDDGDDDDDDGDCDG        Polymer                                    *---------------- ----------------*-----                                      2735  Pol 5'-ATAAAGAAAAAAGACAG     Target                                               TATTTCTTTTTTCTGTC-5'     Duplex                                               GGDDDGGUGDDGGGGCDGUDGUDD Polymer                                    *------------------*-----*-----*-----                                         4956  Pol 5'-GGAAAGGTGAAGGGGCAGTAGTAA                                                                            Target                                               CCTTTCCACTTCCCCGTCATCATT-5'                                                                            Duplex                                     __________________________________________________________________________

In the table, "-" represents a high-specificity base-pair binding, and"*" represents a low-specificity base-pair binding.

The following examples detail synthetic methods for preparing a varietyof subunits, subunit sets, and polymers, in accordance with theinvention. The examples are intended to illustrate but not limit thepresent invention.

EXAMPLE 1 Subunit Protection Methods

A. General Procedure for the Protection of Primary Amino Groups on Basesof Subunits

Unless otherwise indicated, chemicals are purchased from AldrichChemical Co., Milwaukee, Wis.

The subunit, generally a nucleoside or nucleoside analog, (10 mmol,which has been dried by coevaporation with pyridine several times) isdissolved or suspended in pyridine (50-100 mL), and treated withchlorotrimethylsilane (2-3 equivalents of silane per hydroxyl group inthe substrate). The solution is stirred one hour, or until solution iscomplete (sonication may be employed with difficultly solublesubstrates). An alkyl chloroformate, acid chloride, or anhydride, orother suitable activated carboxylic acid derivative is added (1.05-4.0equivalents per amino group in the substrate). After stirring for 1-24hours at room temperature, the reaction is cooled to 0° C., and treatedslowly with a 1:1 mixture of pyridine/water (20 mL). After 10 minutesconcentrated ammonium hydroxide (20 mL) is added and stirring continuedfor 15 minutes. The solution is concentrated under vacuum and dissolvedin ethyl acetate (or ether or chloroform) and shaken with water. Theorganic phase is removed and the product allowed to crystallize. If nocrystallization occurs, the solvent is removed and the residuechromatographed on silica to yield the N-acylated species. Typicalchloroformates which are useful include 9-fluorenylmethoxycarbonylchloride, 2-(p-nitrophenyl)ethoxycarbonyl chloride (Himmelsbach), and2-(phenylsulfonyl)ethoxycarbonyl chloride (Balgobin). Typical acidchlorides include benzoyl, isobutyryl, and trichloroacetyl. Typicalanhydrides include acetic, isobutyric, and trifluroacetic. Other acidderivatives include acyl hydroxybenzotriazolides (prepared from the acidchloride and dry hydroxybenzotriazole in acetonitrile). The latter areadvantageously used to introduce the phenylacetyl group. Alternatively,primary amino groups may be protected as amidines by the procedure ofMcBride, et al.

B. Procedure for the Differential Protection of Primary Diamines onBase-Pair Recognition Moieties

2,6-Diaminopurineriboside (Pfaltz and Bauer, Inc.) is converted by thegeneral procedure in Example 1A into the N-2,N-6bis-(phenylacetyl)amide. The acyl group at the N-6 position isselectively cleaved by treatment of the nucleoside with iN LiOH inpyridine/ethanol at 0° C. The reaction mixture is neutralized with aq.HCl and the solvents evaporated. The residue may be recrystallized fromethyl acetate/ethanol or purified by silica gel chromatography. Thecrude product, or the purified nucleoside, is resubjected to acylationby the general procedure using benzoyl chloride to introduce the N-6benzoyl group. For this second acylation only a slight excess of theacylating agent (1.05-1.2 equivalents) is employed.

C. Procedure for the Protection of Oxo Groups in the RecognitionMoieties

2', 3', 5'-Tri-O-isobutyryl N2-isobutyrl deoxyguanosine is converted bythe procedure of Trichtinger, et al, into the 06 2-(p-nitrophenyl)ethylderivative. Alternatively, guanosine may be converted into the 06diphenylcarbamoyl derivative by the method of Kamimura, et al. Followingtreatment with ammonia (1:1 conc. ammonium hydroxide/DMF) or 1N LiOH inpyridine/ethanol at 0° C., the N2-propionyl 06-diphenylcarbamoylguanosine is produced. These procedures are applicable to thepreparation of N-2 acylated O-4 protected 2-amino-4(3H)-quinazolinonederivatives and N-7 acylated )-9 protected7-amino-9(8H)-imidazo[4,5-f]quinazolinone derivatives.

D. General Procedure for the Introduction of a DimethoxytritylSubstituent at a Primary Alcohol

The alcohol bearing substrate (10 mmol) is dissolved or suspended inpyridine (50-100 mL) and treated with 4,4'-dimethoxytrityl chloride,triethylamine (20 mmol) and 4-dimethylaminopyridine (0.5 mmol). Afterseveral hours at room temperature the mixture is treated with water (5mL) then poured into cold, satd. aq. sodium bicarbonate solution. Themixture is extracted with ethyl acetate (or chloroform) and the combinedorganic layers are dried (sodium sulfate) and evaporated. The residue ischromatographed on silica to give pure dimethoxytritylated compound.

EXAMPLE 2

Preparation of "2-Subunits plus Spacers" Set.

A. Subunits containing 2'-Deoxyribose moiety (FIG. 5A)

The 5'-O-dimethoxytrityl protected derivatives of the following areavailable from Sigma (St. Louis, Mo., USA) : N-4 benzoyldeoxycytidine,N-2 isobutyryldeoxyguanosine, thymidine.2,6-Diaminopurine-2'-deoxyriboside is available from Sigma and isprotected at the primary amino groups and the primary hydroxy group bythe methods in Example 1.

B. Subunits Containing 2'-O-Methylribose Moiety (FIG. 5B)

The 2'-O-methylribonucleosides of uracil, cytosine, guanine, adenine,and 7-deazaadenine may be obtained by the method of Robins, et al (1974)or Sekine, et al. The guanosine and 2-aminoadenosine 2'-O-methyl ethersare also advantageously prepared by the method of Robins, et al, (1981).They may be converted into their base protected analogues by the generalmethods in Example 1 (for example, N-2 isobutyryl for the guanosinederivative, N-2 phenylacetyl, N-6 benzoyl for the 2-aminoadenosinederivative, N-4 benzoyl for the cytidine derivative). The primaryhydroxy is protected as in Example 1.

C. Subunits Containing Morpholino Moiety (FIG. 6A)

A ribose-containing subunit, having the base in the protected form, isoxidized with periodate to a 2'-3' dialdehyde. The dialdehyde is closedon ammonia or primary amine and the 2' and 3' hydroxyls (numbered as inthe parent ribose) are removed by reduction with cyanoborohydride.

An example of this general synthetic scheme is described below withreference to the synthesis of a base-protected cytosine (R_(i) *)morpholino subunit. To 1.6 L of methanol is added, with stirring, 0.1mole of N4-benzoylcytidine and 0.105 mole sodium periodate dissolved in100 ml of water. After 5 minutes, 0.12 mole of ammonium biborate isadded, and the mixture is stirred 1 hour at room temperature, chilledand filtered. To the filtrate is added 0.12 mole of sodiumcyanoborohydride. After 10 minutes, 0.2 mole of toluenesulfonic acid isadded. After another 30 minutes, another 0.2 mole of toluenesulfonicacid is added and the mixture is chilled and filtered. The solidprecipitate is dried under vacuum to give the tosylate salt of the freeamine. The use of a moderately strong (pKa<3) aromatic acid, such astoluenesulfonic acid or 2-naphthalenesulfonic acid, provides ease ofhandling, significantly improved yields, and a high level of productpurity.

Filtration of the tosylate salt of the 2,6-diaminopurine-containingmorpholino subunit also works well. However, the tosylate salts of theguanine-containing and uracil-containing subunits are generally moresoluble in methanol. Thus, for G and U subunits the methanol is removedunder reduced pressure and the residue partitioned between brine andisopropanol--with the desired product going into the organic phase.

The base-protected morpholino subunit can then be protected at theannular nitrogen of the morpholino ring using trityl chloride.

As an example of the tritylation step, to 2 liters of acetonitrile isadded, with stirring, 0.1 mole of the tosylate salt from above, followedby 0.26 mole of triethylamine and 0.15 mole of trityl chloride. Themixture is covered and stirred for 1 hour at room temperature, afterwhich 100 ml of methanol is added, followed by stirring for 15 minutes.The solvent is removed under reduced pressure and then 400 ml ofmethanol is added. After the solid is thoroughly suspended as a slurry,5 liters of water is added, the mixture is stirred for 30 minutes, andfiltered. The solid is washed with 1 liter of water, filtered and driedunder vacuum. The solid is resuspended in 500 ml of dichloromethane,filtered, and rotovaped until precipitation just begins, after which 1liter of hexane is added and stirred for 15 minutes. The solid isremoved by filtering, and dried under vacuum.

The above procedure yields the base-protected morpholino subunittritylated on the morpholino nitrogen and having a free 5' hydroxyl(numbered as in the parent ribose).

D. Subunits Containing N-Carboxymethylmorpholino-5'-amino Moiety (FIG.6C)

A ribose-containing subunit, having the base-pair recognition moiety inthe protected form, is converted to the 5' amine and that 5' aminetritylated, as per Stirchak, Summerton, and Weller (1987), or by themethod described in Example 2E below. Following the general proceduresof Example 2C above, the vicinyl 2' and 3' hydroxyls of the ribose arethen oxidized with periodate to give a 2'-3' dialdehyde. The dialdehydeis closed on glycine in the presence of triethylamine. The 2' and 3'hydroxyls (numbered as in the parent ribose) are subsequently removed byreduction with cyanoborohydride.

Alternatively, the dialdehyde can be closed on ammonia and reduced as inExample 2C, and then the morpholino nitrogen alkylated with bromoaceticacid buffered with N,N-diethylaniline.

These procedures yield the base-protected morpholino subunit having atritylated 5' amine and a carboxymethyl group on the morpholinonitrogen.

E. Subunits Containing N-Carboxymethylmorpholino-alpha(5'-amino) Moiety(FIG. 6F)

Examples 2C and 2D illustrate the preparation of morpholino-containingsubunits wherein the 5' methylene is in the beta orientation--that is,the same orientation as in the parent ribose. Analogousmorpholino-containing subunits wherein the 5' methylene is in the alphaorientation can be prepared by the following general approach.

The 5' hydroxyl of a ribose-containing subunit, having the base-pairrecognition moiety in the protected form, is converted to a secondaryamine by established methods (see Example 2D above). Thereafter,following the general procedures of Example 2C above, the vicinyl 2' and3' hydroxyls of the ribose are oxidized with periodate to give a 2'-3'dialdehyde. The 2' aldehyde rapidly closes on the secondary amine at the5' position (numbered as in the parent Ribose). Reduction withcyanoborohydride then generates a structure containing a morpholino ringwherein the annular morpholino nitrogen is tertiary, and containing a 5'aldehyde in the alpha orientation. Subsequent addition of ammonia or aprimary amine, in the presence of excess cyanoborohydride, generates a5' amine (primary or secondary, respectively) in the alpha orientation.

The above general strategy can be applied to prepare subunits containingN-carboxymethylmorpholino-alpha(5'-amino) moiety, as well as a number ofother useful variations. One method to introduce the desired secondaryamine at the 5' position of the ribose moiety entails: a) conversion ofthe 2', 3' hydroxyls to an acetal as per the method of Smith, Rammler,Goldberg and Khorana (1961); b) oxidation of the 5'hydroxyl to analdehyde using DMSO/pyridine/trifluoroaceticacid/diisoproylycarbodiimide (the Moffat oxidation); c) reacting this 5'aldehyde with glycine (or the tert-Butyl ester of glycine) in thepresence of cyanoborohydride; and, regeneration of the 2', 3' hydroxylsby acid cleavage of the acetal.

F. Subunits Containing Ribose with 5'-Carbazate (FIG. 5C)

A ribose-containing subunit can be converted to the 5'carbazate asfollows. To 10 mMole of ribose-containing subunit, having exocyclicamines of the base-pair recognition moiety in the protected state, add100 ml of anisylaldehyde and 0.5 g of tosic acid. Stir at roomtemperature for 48 hours. Add the reaction mixture to 500 ml hexane andcollect the precipitate. Purify the product by silica gel chromatographydeveloped with ether. The resulting product is reacted with 2equivalents of bis(p-nitrophenyl)carbonate plus 2 equivalents oftriethylamine in acetonitrile for 8 hours at 30° C. The product ispurified by silica gel chromatography developed with a 5% to 15%acetone/chloroform mixture. The product is reacted with 4 equivalents oft-butylcarbazate in DMF for 4 hrs at 50° C. The reaction mixture isadded to water and the precipitate collected and suspended in DMF/ConNH₄ OH, 1:1 by vol overnight at 30° C. The ammonium solution is added tobrine and the insoluble product collected and dried under vacuum. Thedry product is dissolved in trifluoroacetic acid and, after 5 minutes,ether is added to precipitate the product, which is triturated twicewith ether. The product is dissolved in methanol containing sufficientN-ethylmorpholine to neutralize all residual trifluoroacetic acid andthe product again precipitated by addition of ether, and the productdried under vacuum. The desired 5'carbazate product can generally bepurified by silica gel chromatography developed withN-ethylmorpholine/methanol/chloroform, 1:4:6 by volume, or preferably,purified by recrystalization from a suitable aqueous/organic mixture.

G. Subunits Containing Ribose with 5'-Sulfonylhydrazide

A ribose-containing subunit can be converted to the 5'-sulfonylhydrazideas follows. Ten mMole of ribose-containing subunit, having exocyclicamines of the base-pair recognition moiety in the protected state, isconverted to the anisylacetal derivative as described in Example 2Fabove.

To 10 mMole of sulfonyl chloride in dichloromethane chilled on dry iceadd 15 mMole of N,N-diethylaniline. Next, slowly add, with rapidstirring, a dilute solution of 10 mMole of N-aminophthalimide indichloromethane.

After 20 minutes, add the anisylacetal subunit derivative to thischlorosulfonylhydrazide solution. Slowly add, with rapid stirring, 30mMole of diisopropylethylamine in 30 ml of dichloromethane. Afterstirring 1 hour at room temperature, remove the solvent under reducedpressure and purify the product by silica gel chromatography developedwith an acetone/chloroform mixture.

The product is then treated with hydrazine acetate in methanol, thesolvent removed under reduced pressure, and DMF/con NH₄ OH, 1:1 by volis added and the preparation incubated at 30° C. overnight. Lastly, theproduct is treated with trifluoroacetic acid and worked up as in Example2F.

H. Subunits Containing Ribose with 5'-Glycinamide

A primary amine is introduced into the 5' position of aribose-containing subunit following the oxidation/reductive alkylationprocedure described in Example 2E, excepting ammonia, is used instead ofGlycine. This 5' primary amine is then acylated withN-tert-butoxycarbonyl glycine, p-nitrophenyl ester. After purification,the protective groups are removed by treatment with DMF/con NH₄ OH, andthen with trifluoroacetic acid, and the final 5'-glycinamide derivativeworked up as in Example 2F.

I. Subunits containing Ribose with an Aminomethylethylphosphate GroupLinked to the 5'Oxygen

Aminomethylphosphonic acid (Aldrich Chem. Co.) is reacted with tritylchloride in the presence of triethylamine. The di-anionic phosphonateproduct, where the counter ions are triethylammonium, is suspended inethanol and then a carbodiimide, such as dicyclohexylcarbodiimide (DCC),is added. The resultant mono-anionic product is shaken with a mixture ofwater and chloroform containing pyridinium hydrochloride. This proceduregives a mono-ionic phosphonic acid having a pyridinium counter ion. Thisproduct is added to chloroform, followed by addition of theribose-containing subunit wherein exocyclic amines of the base is in theprotected form and the 2' and 3' hydroxyls are protected as theanisylacetal. DCC is added to couple the phosphonate to the 5'oxygen ofthe subunit. The product is dried and chromatographed on silica usingmethanol/chloroform mixtures. The pure product is next base-deprotectedwith DMF/conNH₄ OH, 1:1 by vol. and then suspended in trifluoroaceticacid to remove the trityl and the anisyl protective group

EXAMPLE 3

Preparation of Subunits With Tautomeric Bas

A. Subunit Containing 2'-Deoxyribose Moiety (FIG. 5A)

1. Subunit containing the base of FIG. 13. 4-Acetylamino-2-methylbenzoicacid (Peltier) is converted into the 5-nitro compound by treatment withcold fuming nitric acid. The reaction mix was poured into crushed iceand the solid product collected by filtration and purified byrecrystallization from DMF/water or by silica chromatography. Theacetamide is removed by alkaline hydrolysis with 1-10% NaOH solution in90% ethanol. The reaction mixture was added to excess dilute HCl and thesolvent evaporated. The crude acid is esterified with satd. methanolicHCl at room temperature for several days. After removal of solvent theproduct is partitioned between ethyl acetate and satd. sodiumbicarbonate. After washing with water the organic phase is evaporatedand the residue purified by silica chromatography. The nitro group isreduced to the amino using hydrogen and palladium on carbon in ethanolor DMF. After filtration through celite and evaporation, the crudediamine is converted to the methyl2-amino-6-methylbenzimidazole-5-carboxylate using cyanogen bromide inmethanol at reflux. The mixture is cooled and poured into satd. aq.sodium bicarbonate and the solid product filtered and purified byrecrystallization. The exocyclic amino group is acylated by refluxingwith phthaloyl dichloride in pyridine followed by reaction of thediazepine with pyrazole in refluxing acetonitrile according to themethod of Katritzky. The compound is reacted with either bromine orN-bromosuccinimide or 1,3-dibromo-5,5-dimethylhydantoin either neat orin carbon tetrachloride or chloroform or 1,1,1-trichloroethane with theaid of a high-intensity sun lamp and/or benzoyl peroxide, to provide thebenzylic bromide. It is possible to acylate the diazepine further withisobutryl chloride in pyridine to produce a triply acylatedbenzimidazole species. This is normally done prior to the bromination.

The crude benzylic bromide is reacted with sodium azide in dry DMF andreduced with hydrogen over platinum or palladium to produce the lactam.This is O-silylated with one equivalent of trimethylsilyltrifluoromethanesulfonate or tert-butyldimethylsilyltrifluoromethanesulfonate to produce the O-silyl lactimether/benzimidazole trifluoromethanesulfonate salt. This is reacted with3,5-di-O-toluyl-alpha-D-erythropentofuanosyl chloride (Hoffer) in THF oracetonitrile in the presence of p-nitrophenol by the method of Aoyama togive the protected nucleoside which is purified by silicachromatography. The acyl groups are all removed by a two step procedureinvolving first, hydrazineolysis with hydrazine/ethanol at roomtemperature, then evaporation of solvent and heating the crude residuein refluxing ethanol to fully cleave the phthaloyl residue. Theaminobenzimidazole is protected by reaction with4-(dimethoxymethyl)-morpholine (prepared from 4-formyl morpholine by thegeneral procedure of Bredereck et. al.) in methanol to form the amidine.The remaining reactive site of the benzimidazole is protected byreaction with pivaloyl chloride under the conditions of Example 1.Alternatively, the final acylation may be donewith--(dimethylamino)benzoyl chloride. An alternative amino protectinggroup is formed by reaction of the unprotected benzimidazole with4-(dimethylamino)benzaldehyde in methanol in the presence of piperidine(10 mole %) and methanesulfonic acid (5 mole % ). The resulting imine isacylated as for the amidine. The primary hydroxyl group is protectedwith the dimethoxytrityl group as per Example 1.

2. Subunit containing the base of FIG. 13C. 3-Acetamidophenol (AldrichChemical Co.) is nitrated to give the 2-nitro-5-acetamidophenol.Reduction with hydrogen and palladium/carbon and reaction withtrifluoroacetic anhydride or trichloroacetic anhydride give the2-trihaloacetamido derivative. This is nitrated to give the 4-nitrospecies and the trihaloacetyl group removed by brief ammonolysis to give5-acetamido-2-amino-4-nitrophenol.

2,5-Anhydro-3,4,6-tri-O-benzoyl-D-allonothioamide (Pickering, et al.) istreated with methyl iodide and sodium hydride to give the correspondingmethyl thioimidate. Alternatively the thioamide is reacted withdi-tert-butyl dicarbonate (Aldrich) and 4-dimethylaminopyridine indichloromethane to produce the imide. Alternatively, the imide istreated with methyl iodide or methyl triflate in the presence ofdiisopropylethylamine to give the N-tert-butoxycarbonyl methylthioimidate. Any of these are suitable for reaction with aromatic1,2-diamines or ortho aminophenols to produce benzimidazole orbenzoxazole derivatives of deoxyribosides, respectively.

The aminophenol is reacted with the appropriate activated thioamide fromthe previous paragraph to produce the2-(tri-O-benzoyl-beta-deoxyribosyl)benzoxazole. The N-acetyl andO-benzoyl groups are removed by ammonolysis or hydrazinolysis and thenitro group reduced with hydrogen and palladium/carbon. The aromaticdiamine is reacted with cyanogen bromide in refluxing methanol, and theproduct6-amino-2-(tri-O-benzoyl-betadeoxyribosyl)imidazo[4,5-f]benzoxazolederivative protected as in Example 3A1, and the primary hydroxyprotected as per Example 1.

B. Subunit Containing Ribose Moiety (FIG. 5B)

1. Subunit Containing the Base of FIG. 13B. The ribose nucleoside isprepared as for the deoxyribonucleoside in Example 3A1 except that theO-silylated lactam is reacted in the presence of mercuric bromide orsilver trifluoromethanesulfonate with the ribosyl bromide prepared fromby treatment of 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose with HBrin benzene as per the procedure of Maeba et al.

2. Subunit Containing the Base of FIG. 13C.2,5-Anhydro-3-deoxy-4,6-di-O-toluoyl-D-ribo-hexanothioamide (Pickering,et al.) is converted into the methyl thioimidate, the imide, or theN-tert-butoxycarbonyl methyl thioimidate as in Example 3A2. Any of theseare suitable for reaction with aromatic 1,2-diamines or orthoaminophenols to produce benzimidazole or benzoxazole derivatives ofribosides, respectively.

By the same procedures in Example 3A2, the aminophenol is reacted withthe activated thioamide from the previous paragraph to produce thebenzoxazole which is further converted into the protected nucleoside bythe procedures in Example 3A2.

C. Subunit Containing Morpholino Moiety (FIG. 6A)

1. Subunit Containing the Base of FIG. 13B. The morpholine nucleoside isprepared by reaction of the O-silylated lactam from Example 3A1 withtetraacetyl alpha-D-glucopyranosyl bromide (Sigma) (with or without thepresence of mercuric bromide or silver trifluoromethanesulfonate). Theglycoside is converted into the morpholino nucleoside in the usual wayexcept that twice the normal amount of sodium periodate is employed.Following N-tritylation (Example 2C) and hydrazinolysis of the baseprotecting groups, the base is reprotected as in Example 3A1.

Alternatively, the morpholine nucleoside is prepared by reaction of thebenzylic bromide from Example 3A1 with beta-D-glucopyranosylamine(Tamura, et al.) to give the glycosyl lactam directly. This is convertedinto the morpholino nucleoside by the usual procedure except that twicethe amount of sodium periodate is employed in the oxidation step.Following N-tritylation (Example 2C) and hydrazinolysis of the base,reprotection is accomplished as in Example 3A1.

Alternatively, the methyl 4-acetamido-2-methyl-5-nitrobenzoate fromExample 3A1 is brominated as in Example 3A1 and reacted withbeta-D-glucopyranosylamine. The N-acetyl is removed with 1-10% NaOH in90% ethanol, the nitro is reduced with palladium/carbon and hydrogen,and the aminobenzimidazole is formed by reaction with cyanogen bromidein refluxing ethanol. The aminobenzimidazole is protected as in Example3A1.

Alternatively, the riboside prepared in Example 3B1 is converted into amorpholine-containing subunit following the procedure in Example 2C.This procedure is accomplished prior to deacylation of the phthaloylgroup from the aminobenzimidalole. After morpholine formation andprotection as the N-trityl species, the phthaloyl group is removed as inExample 3A1.

The morpholine nitrogen is protected as the N-trityl by reaction of thefree amine or the tosylate salt with trityl chloride in acetonitrilecontaining triethyamine. The reaction mix is poured into water and thesolid product isolated by filtration and purified by silica gelchromatography.

Subunit Containing the Base of FIG. 13C. By the procedures described inMyers, et al. 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide isconverted into 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl cyanideand then into the corresponding thioamide by the method of Pickering, etal, and then into its activated thioamide derivatives as in Example 3A2.These are suitable for reacting with 1,2-diamines or ortho aminophenolsto produce benzimidazoles or benzoxazole derivatives of galactosides,respectively. A similar procedure may be employed beginning with otherhexose nitriles (Myers, et al.).

By the same procedures in Example 3B2, the aminophenol is reacted withthe activated thioamide from the paragraph above to produce thebenzoxazole which is further converted into the N-protected galactosideby the procedures in Example 3B2. This is converted into the morpholinenucleoside by the usual procedure except that twice the normal amount ofperiodate is employed in the oxidation step. The N-trityl group isintroduced by the method in Example 3C1.

EXAMPLE 4 Preparation of 4-Membered High-Specificity Subunit SetContaining Morpholino Backbone Moieties (FIG. 6A)

A. C:G-specific Subunits

1. Subunit Containing Guanine Base (FIG. 10A). Guanosine is convertedinto its 2-phenylacetyl derivative by the method in Example 1. This isconverted into the morpholine nucleoside tosylate salt by the methods inExample 2C. It may be tritylated by reaction with triphenylmethylchloride in acetonitrile containing triethylamine. The reaction mixtureis poured into water and the product filtered. The product is purifiedby recrystallization from acetonitrile.

2. Subunit containing the base of FIG. 24C. 3-Bromophenol was nitratedto give 5-bromo-2-nitrophenol, then the nitro group was reduced withRaney-Nickel (or hydrogen sulfide/pyridine) and the amine formylatedwith formic acetic anhydride. This was again nitrated and then theoxazole is formed by thermolysis. An alternate method of forming theunsubstituted oxazole ring involves removal of the formamide byammonolysis and treatment of the aminophenol with the formylationreagent derived from phosphorous oxychloride/dimethylformamide. Otheramides than formic may be used for the protection of this amino groupduring the nitraton, and that the oxazole synthesis with these specieswill produce substituted benxoxazoles. For example, thetrifluoracetamide, formed using trifluoroacetic anhydride, will producethe trifluoromethyl benzoxazole. Finally, the nitro group was reducedand the amine protected as a 2-(trimethylsilyl)ethyl carbamate prior tocoupling with the sugar. This product is the key intermediate for theintroduction of the sugar moiety.

The glycosylation procedure employed follows the general method ofZimmermann and Stille for the coupling of vinyl stannanes with aromatichalides, and the method for the conversion of pyranoid glycals to theglucosyl derivatives by the method of Hanessian, et al. The bromideabove (or appropriate aromatic halide in general) was reacted with thepersilylated, C-1 stannylated pyranoid glycal (Hanessian, et al.) in thepresence of tetrakis(triphenylphosphine)palladium(0). The enol ether washydroborated with the borane-dimethyl sulfide complex in tetrahydrofuranand the resulting boron species decomposed to the alcohol by theaddition of basic hydrogen peroxide. All the silylated alcohols andbeta(trimethylsilyl)ethyl carbamates were deprotected with flouride ion.

In this example, the amino group may be reprotected as thetrifluoroacetamide as in Example 1. The molecule was nitrated in aceticacid, the labile amide cleaved by ammonolysis, and theaminobenzimidazole prepared by reduction of the nitro group withhydrogen sulfide/pyridine or stannous chloride with cyanogen bromide.Following protection of the aminobenzimidazole by the methods in Example3A or Example 4Ciii, the morpholine moiety was then constructed as inExample 2C, except that two equivalents of sodium periodate are employedin the oxidation step.

3. Subunit containing the base of FIG. 24D. 2-Bromo-4-methylphenol wasnitrated to produce the 6-nitrospecies. Following reduction withhydrogen sulfide in pyridine, the aminophenol was treated with cyanogenbromide to produce the aminobenzoxazole. Following conversion to the2-(trimethylsilyl)ethyl carbamate, the bromine may be replaced by theappropriate glycosyl unit by coupling with the pyranoid glycal as inExample 4Aii. After the coupling sequence was completed, and themolecule fully desilylated, the amino group was protected as thetrifluoro- or trichloroacetamide, and the molecule nitrated to producethe 4-nitro species. The 5-methyl group was now functionalized byreaction with dimethylformamide dimethylacetal to give the enamine bythe general procedure (Mulzer, et al). Reduction of the nitro group withhydrogen sulfide/pyridine or with hydrogen and Pd/carbon, afforded theindole. As the protecting group on the aminobenzoxazole may be wholly orpartially removed during the indole formation sequence, followingammonolysis to remove any amidine group, the trifluoro- ortrichloroacetamide was reintroduced as in Example 1. Furtherfunctionalization of the indole was done by reaction with phosphorousoxychloride/dimethylformamide (Smith (1954)). The resultant formylindole may be converted into the nitrile by reaction with hydroxylamineto give the oxime followed by treatment with trifluoroacetic anyhdride.The glucose containing subunit may be converted into the morpholinosubunit by the procedure in Example 2C except that two equivalents ofsodium periodate is used.

B. T:A or U:A-Specific Subunits

1. Subunit containing 2,6-diaminopurine base (FIG. 10B).2,6-Diaminopurineriboside is converted into its N2-phenylacetylN6-benzoyl derivative by the method in Example 1. This is converted intothe morpholine nucleoside by the methods in Example 2C. It is tritylatedby the procedure in Example 5A

2. Subunit containing the base of FIG. 26D. 2-Bromo-4-methylphenol wasnitrated to produce the 6-nitrospecies. Following reduction withhydrogen sulfide in pyridine, the aminophenol was treated with cyanogenbromide to produce the aminobenzoxazole. Following conversion to the2-(trimethylsilyl)ethyl carbamate, the bromine may be replaced by theappropriate glycosyl unit by coupling with the pyranoid glycal as inExample 4Aii. After the coupling sequence was completed, and themolecule fully desilylated, the amino group was protected as thetrifluoro- or trichloroacetamide, and the molecule nitrated to producethe 4-nitro species. The 5-methyl group was now functionalized byreaction with dimethylformamide dimethylacetal to give the enamine bythe general procedure (Mulzer, et al). Reduction of the nitro group withhydrogen sulfide/pyridine or with hydrogen and Pd/carbon, afforded theindole. As the protecting group on the aminobenzoxazole may be wholly orpartially removed during the indole formation sequence, followingammonolysis to remove any amidine group, the trifluoro- ortrichloroacetamide was reintroduced as in Example 1. Furtherfunctionalization of the indole was done by reaction with phosphorousoxychloride/dimethylformamide (Smith (1954)). The resultant formylindole may be converted into the nitrile by reaction with hydroxylamineto give the oxime followed by treatment with trifluoroacetic anyhdride.The glucose containing subunit may be converted into the morpholinosubunit by the procedure in Example 2C except that two equivalents ofsodium periodate is used.

3. Subunit containing the base of FIG. 26E. 2-Bromo-4-chlorophenol wasnitrated to yield the 6-nitro species. Following reduction with hydrogensulfide in pyridine, the aminophenol was treated with cyanogen bromideto produce the aminobenzoxazole. Following conversion to the2-(trimethylsilyl)ethyl carbamate, the bromine may be replaced by theappropriate glycosyl unit by coupling with the pyranoid glycal as inExample 4Aii. After the coupling sequence was completed, and themolecule completely desilylated, the amino group is protected as thetrifluoro- or trichloroacetamide as in Example 1, and the chlorineremoved by treatment with triphenyl- or tributyltin hydride and aradical initiator in xylene. The glucose containing subunit may beconverted into the morpholino subunit by the procedure in Example 2Cexcept that two equivalents of sodium periodate is used.

C. A:T or A:U-Specific Subunits

1. Subunit containing the base of FIG. 20B.5-Hydroxy-2(3H)-benzoxazolone (Ozdowska) is acetylated with aceticanhydride and then nitrated with cold fuming nitric acid to the6-nitro-5-acetoxy species. This is dissolved in ethanol and treated withpotassium carbonate, than hydrogenated over palladium to reduce thenitro group to an amino group. The isolated aminophenol is reacted withan active thioamide derivative from Example 3C to give the6-(2,3,4,6-tetra-O-acetylgalactosyl)-oxazolo[4,5-f]-2(3H)-benzoxazolone.Reaction with phosphoryl chloride followed by ammonolysis gives the2-aminobenzoxazole. This is N-protected by the usual procedure toprepare the benzoyl, isobutyryl, acetyl, methoxyacetyl, phenoxyacetyl ortrichloroacetyl amides.

The morpholine nucleoside is prepared from the galactosyl species aboveby the procedures in Example 2C except with double the usual amount ofsodium periodate in the oxidation step in order to form the dialdehydeused for reductive amination. The latter step is performed by the usualmethods. The morpholine is tritylated as in Example 5A and purified bysilica gel chromatography.

2. Subunit containing the base of FIG. 20C. 2-Methyl-4-hydroxybenzoicacid (King) is nitrated with cold fuming nitric acid to give the 5-nitroderivative which is reduced using palladium catalyst in a hydrogenatomosphere to the 5-amino species. This is converted into the methylester by the procedure in Example 3A1. This is converted to the2-aminobenzoxazole using cyanogen bromide and the exocyclic amino groupacylated by the methods in Example 1 with acetyl, methoxyacetyl,trichloroacetyl, isbutyryl or benzoyl. The compound is converted intothe benzylic bromide by the methods in Example 3A1.

The morpholine nucleoside is prepared first by reaction of the benzylicbromide with beta-D-glucopyranosylamine as in Example 3C. Then,methanolic periodate cleavage using twice the usual amount of sodiumperiodate and reductive amination give the morpholine nucleoside. Thisis tritylated by the procedure in Example 5A and purified by silica gelchromatography.

Alternatively, the benzylic bromide is reacted with ammonia to producethe lactam which is O-silylated with trimethylsilyltrifluoromethanesulfonate or tert-butyldimethylsilyltrifluoromethanesulfonate and 2,6-di-tert-butylpyridine. The O-silylatedlactam is reacted with tetraacetyl alpha-D-glucopyranosyl bromide (withour without the presence of silver trifluoromethanesulfonate or mercuricbromide), followed by ammonolysis and reprotection of the primary aminogroup as in Example 5C1. The glycoside is converted into the morpholinenucleoside in the usual way except that twice the normal amount ofsodium periodate is employed. The morpholine is tritylated as in Example5A and purified by silica gel chromatography.

3. Subunit containing the base of FIG. 22B. Alkyl 3-oxobutanoates wereprepared by reaction of an alcohol with diketene. These were convertedto the corresponding alkyl 2-(hydroxyimino)-3-oxobutanoates according tothe general procedure for the ethyl ester (Touster, 1975). This wasreduced and acylated with the corresponding alkyl chloroformate (orrelated acylating species, eg the azidoformate orp-nitrophenylcarbonate) using the general procedure described for theethyl ester in (Fernandez-Resa, et al, 1989), to provide alkyl2-(alkyloxycarbonylamino)-3-oxobutanoates. By these procedures,carbamate derivatives of 2-amino-3-oxobutanoates are available whereinthe ester and carbamate have identical alkyl substituents. By thisdesign, cleavage of the carbamate and ester groups may be performedsimultaneously. Examples of alkyl groups which are available are benzyl(removed by hydrogenolysis), 2-(trimethylsilyl)ethyl (removed byhydrogen fluoride/pyridine), o-nitrobenzyl (removed by photolysis).

To a strirred suspension of sodium hydride (3.0 mmol) in DMF (20 mL)under argon was added benzyl 2-(benzyloxycarbonylamino)-3-oxobutanoate(3.6 mmol) in dimethylformamide (10 mL). The solution of the anion wastreated with 6-chloro-9-(2',3', 5'-tri-O-benzoyl-D-ribofuranosyl)purine(3 mmol, prepared as in Satoda, et al) by the general method of Hamamiciand Miyasaka. The adduct (1 mmol) was dissolved in ethyl acetate/ethanol(20 (mL) containing acetic acid (1 ml) and 5% Pd/carbon (200 mg). Themixture was shaken in a Paar apparatus under 45 psi hydrogen gas. Afterfiltration through celite, the solution was treated with toluenesulfonicacid (1 mmol) and evaporated to dryness. The product was precipitated bydissolving in ethyl acetate/ethanol and pouring into ether/hexanemixtures. This product is also available from other adducts of2-amino-3-oxobutanoates with chloropurines wherein the ester/carbamateis protected with different alkyl groups, some of which are listed inthe preceeding paragraph. For example, if the 2-(trimethylsilyl)ethylgroup is utilized it may be cleaved by reaction in pyridine containinghydrogen fluoride and/or tetrabutylammonium fluoride. The latter reagentmay also be satisfactorily employed in tetrahydrofuran.

The amine tosylate (1 mmol) was dissolved in dimethylformamide (15 mL)and treated with diisopropylamine (3 mmol) and cyanogen bromide (1mmol). The solvent was evaporated, the residue dissolved indichloromethane and washed with sodium bicarbonate solution, water andbrine. After removal of solvent, the residue was redissolved indimethylformamide and treated with 1,8-diazabicyclo[5.4.0]undec-7-ene.The solvent was evaporated and the residue dissolved in dichloromethane,and washed with pH=7 phosphate buffer, water and then dried over sodiumsulfate. Evaporation yields the imidazo[5,1-i]purine which may bepurified by chromatography using chloroform/methanol mixtures.

The imidazo[5,1-i]purine (1 mmol) is dissolved in amethanol/dimethylformamide mixture and treated with 1,3-diaminopropane(20 mmol) at room temperature. After several hours at ambienttemperatures, the mixture was heated in a sealed vessel to complete theremoval of the benzoate esters. Alternatively, the rearrangement to thepyrido[2,3-d:4,5-d']diimidadzole and hydrolysis may be affected with 2Msodium hydroxide in dimethylsulfoxide.

The C-8 oxo group was introduced by the general procedure of Ikehara andKaneko, which involves bromination of the benzimidazole followed by mildhydrolysis with aqueous acetate. This and other tautomericaminobenzimidazole moieties may be protected on the reactive amino andbenzimidazole type nitrogens as was done in Example 3A. Alternatively,the trifluoroacetamide was prepared by using the transient protectionprocedure for alcohols from Example 1 (trimethylsilyl chloride andpyridine) and reacting the silylated species with trifluoroaceticanhydride to acylate the exocylic amino group. A similar reaction schemewill produce the trichlroacetamide. The benzimidazole type ring nitrogenmay then be protected by a following reaction, again using the transientprotection procedure, with p-toluenesulfonyl chloride in pyridine. Inthis latter reaction, two tautomeric sulfonated products may beobtained, but their separation is not essential.

A nontautomeric aminobenzimidazole may be prepared by methylation of theaminobenzimidazole moiety at an appropriate time. For example, thespecies produced in the preceeding paragraph was dissolved in DMSO andtreated with methyl iodide and potassium hydroxide to give themethylated species. This product may also be obtained by the alkylationof the aminobenzimidazole moiety prior to introduction of the oxo group.

The ribose linked species from QB may be converted into the morpholinenucleoside by the procedure in example 2C.

Subunits containing a deoxyribose moiety may be prepared if the initialchloropurine employred is linked to a deoxyribose. Thus, thetri-O-benzoyl derivative of 6-chloropurinedeoxyriboside (prepared bybenzoylation ofthe free hydroxyl species; Robins and Basom (1978)) isemployed for reaction with the aminoacetoacetate.

4. Subunit containing the base of FIG. 30C. 3-Bromophenol was nitratedto produce 5-bromo-2-nitrophenol. This was reduced with hydrogensulfide/pyridine to the aminophenol, and the amino group masked as thetrifluoroacetamide. A second nitration produced the 4-nitrophenolspecies which was treated with ammonia to cleave the trifluoroacetamide.This was reacted with cyanogen bromide to produce the aminobenzoxazolewhich is protected as the trifluoroacetamide. The nitro group wasreduced with pyridine hydrogen sulfide and the amine treated with ethylchloroformate to generate the carbamate. Nitration, nitro reduction, andheating provided the fused imidazolone. Ammonolysis of thetrifluoroacetamide gave the aminobenxoxazole which was protected as thebeta(trimethylsilyl)ethylcarbamate. This was coupled with the pyranoidglycal as in Example 4Aii to produce the subunit with the glucosemoiety. Following complete desilylation, the amino group was protectedas the trifluoro- or trichloroacetamide as per Example 1. The glucosylsubunit may be converted into the morpholine containing subunit by themethod of Example 2C except that two equivalents of sodium periodate isemployed in the oxidation step.

5. Subunit containing the base of FIG. 30F. 3-Bromophenol was nitratedto produce 5-bromo-2-nitrophenol. This was reduced with hydrogensulfide/pyridine or stannous chloride to the aminophenol. This wasreacted with cyanogen bromide to produce the aminobenzoxazole, which wasprotected as the 2-(trimethylsilyl)ethylcarbamate. This was coupled withthe pyranoid glycal as in Example 4Aii to produce the subunit with theglucose moiety. Following complete desilylation of the molecule, theamino group was protected as the trifluoro- or trichloroacetamide as perExample 1. The glucosyl subunit may be converted into the morpholinecontaining subunit by the method of Example 2C except that twoequivalents of sodium periodate is employed in the oxidation step.

D. G:C-Specific Subunits

1. Subunit containing the base of FIG. 15C. 5-Chloro-2,4-dinitrophenol(Carnelley, et al.) is treated with chloromethyl benzyl ether anddiisopropylethyl amine, and the ether is treated with the sodium salt ofmethyl cyanoacetate (or malononitrile) followed by reduction with ironin acetic acid. Cleavage of the acetal (hydrogen/palladium on carbon)and reaction with an activated thioimide derivative from Example 3Cproduces the pyrrolobenzoxazole which, after ammonolysis, may be baseprotected by the procedure in Example 1 to prepare the benzoyl,isobutyryl, acetyl, methoxyacetyl, phenoxyacetyl or trichloroacetylamides.

The morpholine nucleoside is prepared by reaction of the galactosidewith double the usual amount of sodium periodate in order to form thedialdehyde used for reductive amination. The latter step is performed bythe usual methods. The molecule is tritylated by the method in Example5A and purified by silica gel chromatography.

2. Subunit containing the base of FIG. 15B. 4-Chloro-2-methylbenzoicacid (Pfaltz and Bauer Chemical Co.) is converted into its methyl ester(HCl/methanol) and further converted into the benzylic bromide by theprocedure in Example 3C. Reaction with two equivalents of ammoniaprovides the lactam which is nitrated in fuming nitric acid to give the4-nitro-5-chloro-2-oxoisoindole.

The lactam from above is O-silylated as in Example 5C2. The lactim etheris reacted with tetraacetyl alpha-D-glucopyranosyl bromide (with orwithout the presence of silver trifluoromethanesulfonate or mercuricbromide). This is reacted the sodium salt of methyl cyanoacetate (ormalononitrile) followed by reduction with iron in acetic acid. The acylgroups are all removed by ammonolysis and the base reprotected by theusual procedure as the benzoyl, isobutyryl, acetyl, methoxyacetyl,phenoxyacetyl or trichloroacetyl amides.

Alternatively, 4-chloro-2-methylbenzoic acid is nitrated with fumingnitric acid in concentrated sulfuric acid to give the 5-nitroderivative. Following esterification by the method in Example 3A, thisis reacted with the sodium salt of methyl cyanoacetate (ormalononitrile) followed by reduction with iron in acetic acid. The amineis protected by reaction with trichloroacetic anhydride, methoxyaceticanhydride, acetic anhydride, isobutyryl chloride or benzoyl chloride.This is converted into the benzylic bromide by the methods in Example3C. The benzylic bromide is converted into the lactam glucoside bytreatment with beta-D-glucopyranosylamine.

The glucoside above is reacted with methanolic periodate using twice theusual amount of sodium periodate followed by reductive amination to givethe morpholino nucleoside. This is tritylated by the procedure inExample 5A and purified by silica gel chromatography.

3. Subunit containing the base of FIG. 17C. 5-Formyl-2'-deoxyuridine(Barwolff and Langen) is dissolved in methanol and treated withmanganese dioxide in the presence of sodium cyanide and acetic acidaccording to the general procedure of Corey, et al. to provide themethyl ester. The ester is reacted with tert-butyldimethylsilyl triflatein dichloromethane in the presence of diisopropylethyl amine to protectthe alcohols. The heterocycle is activated by the method ofBischofberger (NaH, triisopropylbenzenesulfonyl chloride, THF). The4-O-sulfonated heterocycle is treated with the tosylate salt ofbenzhydryl alanine (Aboderin, et al.) in the presence ofdiisopropylethyl amine in DMF to give the cytosine derivative. Thecytosinyl alanine derivative is oxidized to the dehydroamino acid by thegeneral procedure of Poisel and Schmidt (tert-butyl hypochlorite in THF,followed by one equivalent of potassium tert-butoxide in THF). Theproduct is treated with a catalytic amount of potassium tert-butoxide inhot THF to provide the pyrimidopyridine. The benzhydryl ester is removedby hydrogenolysis using hydrogen over palladium/carbon. The acid istreated with diphenylphosphoryl azide in benzyl alcohol (or benzylalcohol/dioxane) containing triethylamine according to Shioiri, et al.Following hydrogenolysis to cleave the carbamate, and HF-pyridine toremove the silyl groups, the molecule is N-protected as thetrichloroacetamide or phenylacetamide by the usual procedure.

In a similar manner, 5'-formyluridine, prepared from 5-methyluridine bythe procedures in Barwolff and Langen, is converted into thecorresponding pyrimidopyridine riboside. The riboside is converted intothe morpholine nucleoside by the usual procedure, and protected as theN-trityl derivative.

5. Subunit containing the base of FIG. 18B. The imidazo[5,1-i]purine (1mmol) was prepared as in example 4Ciii and dissolved in amethanol/dimethylformamide mixture and treated with 1,3-diaminopropane(20 mmol) at room temperature. After several hours at ambienttemperatures, the mixture was heated in a sealed vessel to complete theremoval of the benzoate esters. Alternatively, the rearrangement andhydrolysis may be affected with 2M sodium hydroxide indimethylsulfoxide.

This and other tautomeric aminobenzimidazole moieties in this disclosuremay be protected on the reactive amino and benzimidazole nitrogens aswas done in Example 3A. Alternatively, the trifluoroacetamide may beprepared by employing the transient protection procedure for alcoholsfrom Example 1 (trimethylsilyl chloride and pyridine) and reacting thesilylated species with trifluoroacetic anhydride to acylate the exocylicamino group. A similar reaction scheme will produce thetrichlroacetamide. The benzimidazole type nitrogen may then be protectedby a following reaction, again using the transient protection procedure,with p-toluenesulfonyl chloride in pyridine. In this latter reaction,two tautomeric sulfonated products may be obtained, but their separationis not essential.

The ribose above may be converted into the morpholine nucleoside by theprocedure in example 2C.

The preparation of subunits containing the deoxyribose moiety can beachieved if the initial chloropurine used contains a deoxyribose. Thus,the tri-O-benzoyl derivative of 6-chloropurinedeoxyriboside (prepared bybenzoylation of the free hydroxyl soecies obtained from Robins andBasom) is employed in the coupling with the aminoacetoacetatederivative.

An alternative entry into compounds of this structural type, whichallows for the placement of substituents at the benzimidazole carbonother than hydrogen, is as follows. 4,6-Dichloro-5-nitropyrimidine wasreacted with beta-1-amino-2,3,4,6-tetra-O-pivaloyl-D-galactose (Kunz andPfrengle). The monoadduct was reduced with Raney-Nickel and hydrogen gasto the 5-amino pyrimidine species. This was converted into the5-acylamino species by reaction with the corresponding acid anhydride oracid chloride, or other acid derivative in pyridine. Thermolysis of theamide (30-200) provided the 6-chloro-8-substituted-9-galactosylpurine.For example, reaction of the amine with difluorothioacetyl fluoride(Martin) yields an intermediate thioamide which was readily cyclized tothe difluoromethyl benzimidazole moiety.

These species may then be treated with an alkyl2-(alkyloxycarbonylamino)-3-oxobutanoate and carried on as above toprepare the galactosyl substituted series. The pivaloyl groups wereremoved in the rearrangement step of the imidazo[5,1-i]purine to thepyrido[2,3-d:4,5-d']diimidazole, as were the benzoyl groups in theribosyl series. Conversion of the final galactosyl substituted subunitinto the morpholine subunits was done as per Example 2C except that 2equivalents of sodium periodate is used.

Other glycosylamines may be employed in the reaction with thedichloronitropyrimidine. Examples include the tetrabenzoates of bothbeta-D-glucosylamine and beta-D-galactosylamine (Caballero, et al).

5. Subunit Containing the Base of FIG. 31C. 4-Chloro-3-nitrophenol wasbrominated to produce the 6-bromo species and reacted withdimethoxymethane and tosic acid in dichloromethane by the method ofYardley and Fletcher to produce the methoxymethyl ether. The ether wasthen treated with the sodium salt of ethyl cyanoacetate in ethanol andthe product reduced with iron filings in acetic acid to provide theamino indole ester. Water was added to produce an 80% acetic acidmixture and the suspension heated. After filtration of the iron andremoval of the solvent, the amino group was protected as the2-(trimethylsilyl)ethyl carbamate by the procedures in Example 1(transient protection). The phenol was nitrated with nitric acid/aceticanhydride, the nitro reduced with hydrogen sulfide/pyridine, and theoxazole formed as in example 4Aii. Examples of substituents which may beincorporated onto the oxazole carbon are hydrogen, methyl,difluoromethyl, and trifluoromethyl. The bromo aromatic was coupled tothe pyranoid glycal to produce the subunit with the glucose moiety.After complete desilylation of the molecule, the amino pyrrole wasprotected as the trifluoro- or trichloroacetamide as per Example 1. Theglucosyl subunit may be converted into the morpholine containing subunitby the method of Example 2C except that two equivalents of sodiumperiodate is employed in the oxidation step.

6. Subunit Containing the Base of FIG. 31D. 3-Fluoro-4-nitrophenol wasreacted with dimethoxymethane and tosic acid in dichloromethane by themethod of Yardley and Fletcher to produce the methoxymethyl ether. Theether was the treated with the sodium salt of ethyl cyanoacetate inethanol and the product reduced with iron filings in acetic acid toprovide the amino indole ester. Water was added to produce an 80% aceticacid mixture and the suspension is heated. After filtering the iron, andremoval of the solvent, the amino group was protected as the2-(trimethylsilyl)ethyl carbamate by the procedures in Example 1(transient protection). The phenol was reacted withtrifluoromethanesulfonic anhydride and the triflate coupled to thepyranoid glycal by the method in Example 4Aii to produce the subunitwith the glucose moiety. Following complete desilylation of themolecule, the amino pyrrole was protected as the trifluoro- ortrichloroacetamide as per Example 1. The glucosyl subunit may beconverted into the morpholine containing subunit by the method ofExample 2C except that two equivalents of sodium periodate is employedin the oxidation step.

EXAMPLE 5 Preparation of 4-Membered High-Specificity Subunit SetContaining N-Carboxymethylmorpholino-5'-amino Backbone (FIG. 6C)

Subunits containing ribose, galactose, or glucose moieties are preparedas in Example 4, and their respective sugar moieties are converted tothe N-Carboxymethylmorpholino-5'-tritylated amine form by the methoddescribed in Example 2D.

EXAMPLE 6 Polymer Assembly Procedures for 2'-O-Methylribose (FIG. 5B)and 2'-Deoxyribose-containing (FIG. 5A) subunits

The protected 2'-Deoxyriboside-containing subunits and the protected2'-O-Methylriboside-containing subunits are converted into theircorresponding 3'-H-phosphonate salts by the methods given in Sakataume,et al. and polymerized on solid support by the method in this source.When the assembly of the polymer chain is complete, the supportedmolecule is treated with a primary or secondary amine in the presence ofeither iodine or carbon tetrachloride as per the method of Froehler. Thephorphoramidate-linked polymer is removed from the support anddeprotected by the usual methods involving ammonolysis (Froehler).

EXAMPLE 7 Representative Activation Procedures for Morpholino-ContainingSubunits

A. Activation of 5'-Hydroxyl of Morpholino Subunit

Dimethylaminodichlorophosphate is prepared as follows: a suspensioncontaining 0.1 mole of dimethylamine hydrochloride in 0.2 mole ofphosphorous oxychloride is refluxed for 12 hours and then distilled(boiling point is 36° C. at 0.5 mm Hg).

Activation of the 5'Hydroxyl of a morpholino-containing subunit preparedas in Example 2C entails dissolving one mmole of 5'hydroxyl subunit,baseprotected and tritylated on the morpholino nitrogen, in 20 ml ofdichloromethane. To this solution 4 mmole of N,N-diethylaniline and 1mmole of 4-methoxypyridine-N-oxide are added. After dissolution, 2 mmoleof dimethylaminodichlorophosphate is added. After two hours the productis isolated by chromatography on silica gel developed with 10%acetone/90% chloroform. The same procedure, except substitutingethyldichlorothiophosphate instead of dimethylaminodichlorophosphate,gives an activated subunit with similar utility.

B. Activation of 5'-Amine of Morpholino-Containing Subunit

The 5'hydroxyl of a morpholino-containing subunit, having exocyclicamino groups of the base-pair recognition moiety in the protected form,prepared as in Example 2C can be converted to the amine as follows. To500 ml of DMSO is added 1.0 mole of pyridine (Pyr), 0.5 mole oftriflouroacetic acid (TFA), and 0.1 mole of the morpholino subunit. Themixture is stirred until dissolved, and then 0.5 mole ofdiisopropylcarbodiimide (DIC) or dicyclohexylcarbodiimide (DCC) isadded. After 2 hours the reaction mixture is added to 8 liters ofrapidly stirred brine, which is stirred for 30 minutes and filtered. Thesolid is dried briefly, washed with 1 liter of ice cold hexanes,filtered, and the solid is added to 0.2 mole of sodium cyanoborohydridein 1 liter of methanol, stirred for 10 minutes, 0.4 mole ofbenzotriazole or p-nitrophenol is added, followed by 0.2 mole ofmethylamine (40% in H₂ O) and the preparation is stirred four hours atroom temperature [Note: the benzotriazole or p-nitrophenol buffers thereaction mixture to prevent racemization at the 4' carbon of the subunitat the iminium stage of the reductive alkylation]. Finally, the reactionmixture is poured into 5 liters of water, stirred until a goodprecipitate forms, and the solid is collected and dried. This driedproduct is next suspended in DMF and 4 equivalents of SO₃ /pyridinecomplex is added. Over a period of several hours, 8 equivalents oftriethylamine is added dropwise with stirring. After an additional twohours the preparation is dumped into a large volume of brine and thesolid collected by filtration and dried. This sulfamic acid preparationis then purified by silica gel chromatography.

Ten mmole of the triethylamine salt of sulfated subunit protected on therecognition moiety and on the nitrogen of the morpholino ring isdissolved in 10 ml of dichloromethane and then 40 mmole of pyridine isadded. This solution is chilled for 15 minutes on a bed of dry ice andthen 1.1 mmole of phosgene (20% in Toluene) is slowly added while thesolution is rapidly stirred. After addition, the solution is allowed tocome to room temperature and then washed with aqueous NaCHO₃,dried, andchromatographed on silica gel eluted with a mixture of chloroform andacetone to give the desired sulfamoyl chloride.

C. Activation of Annular Morpholino Nitrogen

This example describes the preparation of a morpholino subunit protectedon its 5' oxygen and sulfated on its morpholino ring nitrogen.Morpholino-containing subunit prepared as in Example 2C, but not carriedthrough the last tritylation step, is silylated on its 5' hydroxyl witht-butyldimethlsilyl chloride. This product is then treated with SO₃/pyridine complex (with excess pyridine) in dimethylformamide (DMF) togive a sulfamic acid on the annular morpholino nitrogen.

It should be mentioned that the salts of sulfamic acids can bechromatographed on silica gel using triethylamine/methanol/chloroformmixtures if the silica is first pre-eluted with 2% triethylamine inchloroform.

This sulfamic acid on the morpholino nitrogen is converted to thesulfamoyl chloride and purified as in Example 7B above.

D. Activation of N-Carboxymethyl of Morpholino

Carboxylate-containing subunits, such as prepared in Examples 2D and 2E,are activated as follows. Ten mmole of the subunit is dissolved in DMFcontaining 20 mmole of p-nitrophenol and 15 mmole ofdicyclohexylcarbodiimide. After 1 hour the product is rotovaped and thenpurified by silica gel chromatography developed with a mixture ofacetone and chloroform.

EXAMPLE 8 Representative Solid-Phase Polymer Assembly ofMorpholino-containing Subunits (FIG. 33)

This example describes a method which is generally applicable forassembly of activated subunits, prepared as in Examples 7A and 7B, togive phosphorodiamidate-linked, ethylthiophosphoramidate-linked, andsulfamate-linked binding polymers. A similar scheme wherein the couplingstep includes the addition of silver trifluoromethanesulfonate, and useof N,N-diisopropyl-2-methoxyethylamine instead ofdiisopropylethanolamine, is suitable for assembly of subunits preparedas in Example 7C to give sulfamate-linked polymers. A similar scheme,wherein the coupling step is carried out in dimethylformamide instead ofdichloromethane, is suitable for assembly of subunits activated as inExample 7D to give amide-linked polymers.

A. Linker

Aminomethyl polystyrene resin (Catalog no. A1160, from Sigma ChemicalCo.) 1% divinylbenzene crosslinked, 200 to 400 mesh, 1.1 mMole of N pergram, is suspended in dichloromethane and transferred to a 1 cm diametercolumn having a frit on the bottom, to give a resin bed volume of 2.5ml.

One mMole of bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (PierceChemical Co. of Rockford, Ill. USA) is added to a dichloromethanesolution containing 1 mMole of N-tritylated piperazine. After 2 hoursthe reaction mixture is chromatographed on silica gel developed with anacetone/chloroform mixture to give a mono-activatedbeta-elimination-cleavable linker.

134 MicroMole of the above linker is dissolved in 1 ml ofdichloromethane and added to the resin in the synthesis column and theresin suspension agitated for 3 hours at 30° C. Next, 1 mMole ofdiisopropylaminoethanol and 1 mMole of acetic anhydride is added andagitation continued for 10 minutes, followed by addition of 2 mMole ofbenzylmethylamine and agitation for 20 minutes. The column is washedwith 30 ml dichloromethane. Based on release of trityl, the aboveprocedure typically gives on the order of 100 to 110 micromoles of boundlinker.

B. Coupling Cycle (Detritylation/Coupling/Capping)

The coupling cycle described below is used for adding each subunit in anorder appropriate to give a polymer having the desired sequence ofsubunits.

1. Detritylation. Add a solution containing 53 ml of dichloromethane, 6ml of trifluoroethanol, and 1 gram of cyanoacetic acid. After thissolution has passed through, wash the column with 40 ml ofdichloromethane, followed by 20 ml of dichloromethane containing 4 mMoleof diisopropylaminoethanol. Wash the column with 10 ml ofdichloromethane.

2. Coupling. Add 1 ml of dichloromethane containing 120 microliter ofdiisopropylaminoethanol to 0.25 mMole of activated subunit (prepared asin Example 7A or 7B) and add to the column and agitate at 37° C. for 1hr. Wash the column with 30 ml dichloromethane. Note: excess unreactedactivated subunit can be conveniently recovered simply by adding 4volumes of hexane to this eluant and filtering.

3. Capping. Add to the column 2 ml of dichloromethane containing 1 mMoleof diisopropylaminoethanol and 1 mMole of acetic anhydride and agitateat 37° C. for 10 minutes. Add to the column 10 ml of dichloromethanecontaining 1 mMole of benzylmethylamine, and agitate the resin bed at37° C. for 20 min. Wash the column with 30 ml dichloromethane.

C. Cleavage from support and deprotection

After all the subunits have been added by the above coupling procedure,the full length polymer is cleaved from the support by eluting thecolumn with a solution consisting of 2.5 ml of diethylmalonate, 5 ml of1,8-diazabicyclo[5.4.0]undec-7-ene, and 43 ml dichloromethane. Thepolymer is then precipitated from this eluant by adding ether.

If it is desirable to add a moiety to enhance aqueous solubility, or toenhance target binding affinity, or to facilitate uptake by specificcell or tissue types, then the secondary aliphatic amine generated uponcleavage from the polystyrene support provides a site for attachment ofsuch moieties at this stage of the polymer preparation.

The polymer product is next dissolved in DMF and an equal volume ofconNH₄ OH added, the preparation capped tightly, and incubated 18 hrs at37° C. Subsequently, the preparation is dried under reduced pressure togive a polymer preparation wherein the base-pair recognition moietiesare deprotected and at one end of the polymer is a trityl moiety, and atthe other end is a secondary aliphatic amine which, as noted above, maybe derivatized prior to the ammonia treatment.

EXAMPLE 9 Polymer Purification Methods

The full-length polymer having a terminal trityl moiety (typicallygreater than 50% of the total mass of the preparation for a 24-subunitlong polymer) can be separated from the capped failure sequences by lowpressure chromatography on a column of chromatographic gradepolypropylene (Catalog No. 4342 from PolySciences Inc.) developed withan acetonitrile/water gradient, with the eluant monitoredphotometrically at 254 nm. Purifications are typically performed usingthe following conditions: the polymer is suspended in water, thesolution adjusted to pH 11 with dimethylamine and the eluting solventsalso adjusted to pH 11 with dimethylamine. In this system, thetritylated full-length polymer elutes appreciably later than thenon-trityl-containing capped failure sequences.

The fractions containing full-length polymer are collected and drieddown under reduced pressure. The polymer preparation is thendetritylated by suspending in trifluoroethanol (1 g polymer in 25 mlTFE) and 1.5 ml of mercaptoacetic acid added. After 10 minutes, 100 mlof ether is added and the final pure product collected by centrifugationor filtration.

EXAMPLE 10 Polymer Assembly Via Novel Oxidation/Ring Closure/ReductionMethod (FIG. 39)

A. Synthesis Support

The solid support used in this synthesis should be hydrophilic, butshould not contain vicinyl hydroxyls. Add an aqueous slurry of"MACRO-PREP 50 CM" (Catalog No. 156-0070 from Bio-Rad Laboratories,Richmond, Calif., USA) to a fritted column to give a 5 ml packed bedvolume (containing approximately 1 mMole of carboxylate). Wash thissynthesis support with 100 ml of 0.1N HCl and then 50 ml water. Pass 50ml of DMF (dimethylfoyrmamide) through the column and drain. Add 5 ml ofDMF containing 5 mMole of diisopropylcarbodiimide and 5 mMole ofp-nitrophenol and incubate with agitation at 30° C. for 3 hours. Washthe column with 100 ml of DMF and then add 20 mMole of piperazine in 10ml of DMF and agitate for 15 minutes. Wash the column with 50 ml of DMFand drain.

B. Addition of Linker and First Subunit

To 1 mMole of a ribose-containing subunit having a carbazate moiety atthe 5' of the ribose (prepared as in Example 2F) in 5 ml of DMF, add 3mMole of Bis[2-(succinimidooxycarbonyloxy)-ethyl]sulfone (PierceChemical Co. of Rockford, Ill., USA) and incubate at 30° C. for 3 hours.To the reaction mixture add ether and collect the precipitate. Wash theprecipitated linker-subunit with ether, resuspend in 5 ml of DMF, add tothe synthesis support, and incubate with agitation for 3 hrs at 30° C.Wash the support with 50 ml of DMF, and then with 100 ml of water.

C. Coupling Cycle

1. Oxidation of Vicinyl Hydroxyls. Dissolve 5 mMole of sodium periodatein 10 ml of water, add to column, and agitate for 10 minutes. Washcolumn with 50 ml of water and drain.

2. Morpholino Ring Closure/Reduction. Dissolve 2 mMole of sodiumcyanoborohydride in 5 ml of water, adjust pH to between 7 and 8 withtrimethylacetic acid, add 1.5 mMole of the next ribose-containing5'-carbazate subunit, and add to the column containing the synthesissupport. Incubate with agitation for 30 min at 30° C. Add formic acid toreduce pH to between 3 and 4, and incubate at 30° C. for 10 minutes.Wash column with 100 ml of water.

Repeat this coupling cycle until all subunits have been added to givethe desired full-length polymer.

3. Addition of Terminal Moieties. If it is desirable to add to thebinding polymer a moiety to enhance aqueous solubility, or to enhancetarget binding affinity, or to facilitate uptake by specific cell ortissue types, this can be conveniently achieved at this stage byoxidizing the vicinyl hydroxyls of the terminal subunit of the polymerand, by the morpholino ring closure/reduction procedure described above,adding said moieties containing a primary aliphatic amine.

4. Cleavage from the Support. After all the subunits of the polymer, andany desired additional groups, have been added by the above couplingprocedure, the polymer is cleaved from the support by washing the columnwith 50 ml of DMF, and then eluting the column with a solutionconsisting of 2.5 ml of diethylmalonate, 5 ml of1,8-diazabicyclo[5.4.0]undec-7-ene, and 43 ml of DMF. The polymer isthen precipitated from this eluant by adding ether.

The full-length polymer can be purified by low pressure chromatographyon a column of chromatographic grade polypropylene (Catalog No. 4342from PolySciences Inc.) developed with an acetonitrile/water gradient,with the eluant monitored photometrically at 254 nm. Purificationsgenerally go better when the polymer is suspended in water and then thesolution adjusted to pH 11 with dimethylamine and the eluting solventsalso adjusted to pH 11 with dimethyl amine.

Although the invention has been described with respect to particularpolymer subunits, methods of preparing the subunits, and polymerassembly, it will be appreciated that various modifications and changesmay be made without departing from the invention.

It is claimed:
 1. A polymer composition effective to bind in asequence-specific manner to a target sequence of a duplex polynucleotidecontaining at least two different-oriented Watson/Crick base-pairs atselected positions in the target sequence, comprising a specificsequence of subunits having the form: ##STR16## where Y is a 2- or3-atom length, uncharged intersubunit linkage group; R' is H, OH, orO-alkyl; the 5'-methylene has a β stereochemical orientation in the5-membered ring and a uniform stereochemical orientation in the6-membered ring; R_(i) has a β stereochemical orientation; and at leastabout 70% of R_(i) groups in the polymer are selected from two or moreof the following base-pair-specificity groups:(a) for a T:A or U:Aoriented base-pair, R_(i) is selected from the group consisting ofplanar bases having the following skeletal ring structures and hydrogenbonding arrays, where B indicates the polymer backbone: ##STR17## wherethe * ring position may carry a hydrogen-bond donor group, such as anamine; and X may be a moiety effective to afford discriminative bindingon the basis of the moiety at the 5 position of the pyrimidine of thetarget base-pair; (b) for a C:G oriented base-pair, R_(i) is selectedfrom the group consisting of planar bases having the following skeletalring structures and hydrogen bonding arrays, where B indicates thepolymer backbone: ##STR18## where the * ring position may carry ahydrogen-bond acceptor group, such as a carbonyl oxygen; X may be amoiety effective to afford discriminative binding on the basis of themoiety at the 5 position of the pyrimidine of the target base-pair; andZ is oxygen or sulfur; (c) for a G:C oriented base-pair, R_(i) isselected from the group consisting of planar bases having the followingskeletal ring structures and hydrogen bonding arrays, where B indicatesthe polymer backbone: ##STR19## where the * ring position may carry ahydrogen-bond acceptor group, such as a carbonyl oxygen; and X may be amoiety effective to afford discriminative binding on the basis of themoiety at the 5 position of the pyrimidine of the target base-pair; and,(d) for an A:T or A:U oriented base-pair, R_(i) is selected from thegroup consisting of planar bases having the following skeletal ringstructures and hydrogen bonding arrays, where B indicates the polymerbackbone: ##STR20## where the * ring position may carry a hydrogen-bonddonating group, such as NH₂ ; and X may be a moiety effective to afforddiscriminative binding on the basis of the moiety at the 5 position ofthe pyrimidine of the target base-pair.
 2. The polymer composition ofclaim 1, containing one or more subunits of the form: ##STR21##
 3. Thepolymer composition of claim 1, containing one or more subunits of theform: ##STR22##
 4. The polymer composition of claim 3, containing one ormore subunits of the form: ##STR23##
 5. The polymer composition of claim1, for use in sequence-specific binding to a B-form DNA-DNA duplexnucleic acid, wherein the Y linkage groups is three atoms in length. 6.The polymer composition of claim 5, wherein one or more subunits of thepolymer are selected from the group consisting of: ##STR24##
 7. Thepolymer composition of claim 6, wherein one or more subunits of thepolymer are selected from the group consisting of: ##STR25##
 8. Thepolymer composition of claim 1, for use in sequence-specific binding toan A-form duplex nucleic acid, wherein the Y linkage group is two atomsin length.
 9. The polymer composition of claim 8, wherein one or moresubunits of the polymer are selected from the group consisting of:##STR26##
 10. The polymer composition of claim 8, wherein one or moresubunits of the polymer are selected from the group consisting of:##STR27##
 11. The polymer composition of claim 1, which contains atleast one R_(i) structure selected from the group consisting of:##STR28##
 12. The polymer of claim 1, wherein at least one R_(i)structure specific for a G:C oriented base pair is selected from thegroup consisting of the following bases: ##STR29##
 13. The polymer ofclaim 1, wherein at least one R_(i) structure specific for an A:T or A:Uoriented base-pair is selected from the group consisting of thefollowing bases: ##STR30##
 14. The polymer of claim 1, wherein at leastone R_(i) structure specific for a C:G oriented base-pair is selectedfrom the group consisting of the following bases: ##STR31##
 15. Thepolymer of claim 1, wherein at least one R_(i) structure specific for anT:A or U:A oriented base-pair is selected from the group consisting ofthe following bases: ##STR32##
 16. The polymer composition of claim 1,wherein up to about 30% of the R_(i) groups in the polymer are cytosine,at polymer subunits corresponding to a G:C oriented base-pair in thetarget sequence, and thymine or uracil, at polymer subunitscorresponding to A:T or A:U oriented base-pair in the target sequence.17. The composition of claim 1, wherein the polymer contains one or moreattached moieties effective to enhance the solubility of the polymer inaqueous medium.
 18. A method for coupling a first free orpolymer-terminal subunit having one of the following subunit forms:##STR33## where R_(i) is a planar ring structure having two or morehydrogen-bonding sites, with a second free or polymer-terminal subunithaving one of the following subunit forms: ##STR34## where Z is a 2-atomor 3-atom long moiety, said method comprising: i) oxidizing the firstsubunit to generate a dialdehyde intermediate;ii) contacting thedialdehyde intermediate with the second subunit under conditionseffective to couple a primary amine to a dialdehyde; and (iii) adding areducing agent effective to give a coupled structure selected from thefollowing forms: ##STR35##
 19. A method for isolating, from a liquidsample, a duplex nucleic acid having a selected target sequence ofbase-pairs, comprising:i) contacting the sample with a polymer reagentcontaining a structure which allows isolation of the reagent fromsolution, and attached to this structure, the polymer composition ofclaim 1 having a subunit sequence effective to bind in asequence-specific manner with the selected target sequence ofbase-pairs, under conditions effective for sequence-specific binding ofthe polymer composition to the selected target sequence of base-pairs;and ii) separating the polymer reagent from the fluid sample.
 20. Themethod of claim 19, for use in detecting the presence of such targetfragment in a liquid sample, which further includes testing theseparated polymer reagent for the presence of bound duplex nucleic acid.21. The method of claim 20, wherein said polymer reagent includes asolid support with bound polymer composition, and said testing includesadding to the duplex nucleic acid, a fluorescent compound effective tointercalate into duplex DNA.
 22. A subunit composition for use informing a polymer composition effective to bind in a sequence specificmanner to a target sequence in a duplex polynucleotide, comprising oneof the following subunit structures: ##STR36## where R' is H, OH, orO-alkyl; the 5'-methylene has a β stereochemical orientation in subunitforms (a), (c), and (d) and a uniform stereochemical orientation insubunit form (b); X is hydrogen or a protective group or a linking groupsuitable for joining the subunits in any selected order into a linearpolymer; Y is a nucleophilic or electrophilic linking group suitable forjoining the subunits in any selected order into a linear polymer; and Xand Y together are such that when two subunits of the subunit set arelinked the resulting intersubunit linkage is 2 or 3 atoms in length anduncharged; Z is a 2-atom or 3-atom long moiety; and, R_(i), which may bein the protected state and has a β stereochemical orientation, isselected from the group consisting of planar bases having the followingskeletal ring structures and hydrogen bonding arrays, where B indicatesthe aliphatic backbone moiety: ##STR37## where the * ring position maycarry a hydrogen-bond acceptor group; and X may be a moiety effective toafford discriminative binding on the basis of the moiety at the 5position of the pyrimidine of the target base-pair; or, where R_(i) isselected from the group consisting of planar bases having the followingskeletal ring structures and hydrogen bonding arrays, where B indicatesthe aliphatic backbone moiety: ##STR38## where the * ring position maycarry a hydrogen-bond donating group; and X may be a moiety effective toafford discriminative binding on the basis of the moiety at the 5position of the pyrimidine of the target base-pair.
 23. A subunitcomposition for use in forming a polymer composition effective to bindin a sequence specific manner to a target sequence in a duplexpolynucleotide, comprising one of the following subunit structures:##STR39## where the 5'-methylene has a β stereochemical orientation insubunit forms (b), and (c) and a uniform stereochemical orientation insubunit form (a); X is hydrogen or a protective group or a linking groupsuitable for joining the subunits in any selected order into a linearpolymer; Y is a nucleophilic or electrophilic linking group suitable forjoining the subunits in any selected order into a linear polymer; and Xand Y together are such that when two subunits of the subunit set arelinked the resulting intersubunit linkage is 2 or 3 atoms in length anduncharged; Z is a 2-atom or 3-atom long moiety; and, R_(i), which may bein the protected state and has a β stereochemical orientation, isselected from the group consisting of planar bases having the followingskeletal ring structures and hydrogen bonding arrays, where B indicatesthe aliphatic backbone moiety, the starred atom is not a hydrogen bondacceptor, and W is oxygen or sulfur: ##STR40##
 24. A polymer compositioncapable of discriminating, on the basis of binding affinity, between (a)a selected target sequence in a duplex polynucleotide containing atleast one pyrimidine-purine base-pair at a selected position in thetarget sequence, wherein the pyrimidine base has a hydrogen on the 5ring position, and (b) the same sequence but in which the primidine ofthe base pair at the selected position contains a methyl group on the 5ring position of the pyrimidine, said composition comprisinga polymerhaving the repeating unit ##STR41## where B is a backbone moiety andwhere at least one R_(i) is selected from the group consisting of##STR42## where X has a size and shape effective to selectively reducebinding of R_(i) to a base-pair having a methyl on the 5 ring positionof the pyrimidine.25.
 25. The polymer of claim 24, wherein at least oneR_(i) is selected from the group consisting of (a), (b), (c), (d), (e)and (f), and where X is selected from the group consisting of sulfur,chlorine, bromine, cyano, azido, difluoromethyl and trifluoromethyl. 26.The polymer of claim 24, wherein at least one R_(i) is selected from thegroup consisting of (g) and (h), and where X is selected from the groupconsisting of oxygen, fluorine, and methyl.